Compositions and methods for inhibiting expression of a gene from the jc virus

ABSTRACT

The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a gene from the JC Virus (JC virus genome), comprising an antisense strand having a nucleotide sequence which is less that 30 nucleotides in length, generally 19-25 nucleotides in length, and which is substantially complementary to at least a part of a gene from the JC Virus. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases caused by JC virus expression and the expression of a gene from the JC Virus using the pharmaceutical composition; and methods for inhibiting the expression of a gene from the JC Virus in a cell.

RELATED APPLICATIONS

This application is a continuation of co-pending U.S. application Ser.No. 11/741,205, filed Apr. 27, 2007 (now U.S. Pat. No. ______) andclaims the benefit of U.S. Provisional Application No. 60/795,765, filedApr. 28, 2006; both applications are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

This invention relates to double-stranded ribonucleic acid (dsRNA), andits use in mediating RNA interference to inhibit the expression of oneof the genes of the JC virus and the use of the dsRNA to treatpathological processes mediated by JC virus infection, such as PML.

BACKGROUND OF THE INVENTION

Progressive multifocal leukoencephalopathy (PML) is a fataldemyelinating disease of the central nervous system which results fromreactivation of the latent polyomavirus JC virus (JCV) and itsproductive replication in glial cells of the human brain (Berger, J. R.(1995) J. Neurovirol. 1:5-18). Once a rare disease primarily seen inpatients with impaired immune systems due to lymphoproliferative andmyeloproliferative disorders, PML has become one of the major neurologicproblems among patients with AIDS (Cinque, P., (2003). J. Neurovirol.9(Suppl. 1):88-92).

It has been reported that between 4 and 8% of AIDS patients exhibitsigns of PML, and JCV has been detected in the cerebrospinal fluid ofaffected patients, suggesting that there is active replication of thevirus in the brain (Berger, J. R. (1995) J. Neurovirol. 1:5-18,Clifford, D. B., (2001) J. Neurovirol. 4:279). In addition, PML hasrecently been seen in patients undergoing experimental treatment withTsybari, an anti VLA4 antobody, in combination with interferon. Thehistological hallmarks of PML include multifocal demyelinated lesionswith enlarged eosinophilic nuclei in oligodendrocytes and enlargedbizarre astrocytes with lobulated hyperchromatic nuclei within whitematter tracts of the brain (Cinque, P., (2003). J. Neurovirol. 9(Suppl.1):88-92), although in some instances atypical features that include aunifocal pattern of demyelination and involvement of the gray matterhave been reported (Sweeney, B. J., (1994). J. Neurol. Neurosurg.Psychiatry 57:994-997). Earlier observations from in vitro cell culturestudies and an in vivo evaluation of JCV in clinical samples led toearly assumptions that oligodendrocytes and astrocytes are the onlycells which support productive viral infections (Gordon, J. (1998) Int.J. Mol. Med. 1:647-655). Accordingly, molecular studies have providedevidence for cell-type-specific transcription of the viral early genomein cells derived from the central nervous system (Raj, G. V., (1995)Virology 10:283-291). However, subsequent studies have shown low, butdetectable, levels of JCV gene expression in nonneural cells, includingB cells, and noticeably high levels of production of the viral earlyprotein in several neural and nonneural tumor cells in humans (Gordon,J. (1998) Int. J. Mol. Med. 1:647-655, Khalili, K., 2003. Oncogene22:5181-5191).

Like the other polyomaviruses, JCV is a small DNA virus whose genome canbe divided into three regions that encompass the transcription controlregion; the genes responsible for the expression of the viral earlyprotein, T antigen; and the genes encoding the viral late proteins, VP1,VP2, and VP3. In addition, the late genome is also responsible forproduction of an auxiliary viral protein, agnoprotein. T-antigenexpression is pivotal for initiation of the viral lytic cycle, as thisprotein stimulates transcription of the late genes and induces theprocess of viral DNA replication. Recent studies have ascribed animportant role for agnoprotein in the transcription and replication ofJCV, as inhibition of its production significantly reduced viral geneexpression and replication (M. Safak et al., unpublished observations).Furthermore, the agnoprotein dysregulates the cell cycle by altering theexpression of several cyclins and their associated kinases (Darbinyan,A., (2002) Oncogene 21:5574-5581).

Thus far, there are no effective therapies for the suppression of JCVreplication and the treatment of PML. Cytosine arabinoside (AraC) hasbeen tested for the treatment of PML patients, and the outcome in someinstances revealed a remission of JCV-associated demyelination (Aksamit,A. (2001) J. Neurovirol. 7:386-390). Reports from the AIDS ClinicalTrial Group Organized Trial 243, however, have suggested that there isno difference in the survival of human immunodeficiency virus type 1(HIV-1)-infected patients with PML and that of the control population,although in other reports it has been suggested that the failure of AraCin the AIDS Clinical Trial Group trial may have been due to insufficientdelivery of the AraC via the intravenous and intrathecal routes (Levy,R. M., (2001) J. Neurovirol. 7:382-385). Based on in vitro studiesshowing the ability of inhibitors of topoisomerase to suppress JCV DNAreplication, the topoisomerase inhibitor topotecan was used for thetreatment of AIDS-PML patients, and the results suggested that topotecantreatment may be associated with a decreased lesion size and prolongedsurvival (Royal, W., III, (2003) J. Neurovirol. 9:411-419).

Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). WO 99/32619 (Fire et al.) discloses the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofgenes in C. elegans. dsRNA has also been shown to degrade target RNA inother organisms, including plants (see, e.g., WO 99/53050, Waterhouse etal.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D.,et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanismhas now become the focus for the development of a new class ofpharmaceutical agents for treating disorders that are caused by theaberrant or unwanted regulation of a gene.

Recent reports have indicated that in vitro, RNAi may show some promisein reducing JC virus replication (Radhakrishnan, S. (2004) J. Vir.78:7264-7269, Orba, Y. (2004) J. Vir. 78:7270-7273). However, the RNAiagents examined were not designed against all know JC Virus strains andwere not selected for stability and other properties need for in vivotherapeutic RNAi agents. Accordingly, despite significant advances inthe field of RNAi, there remains a need for an agent that canselectively and efficiently silence a gene in the JC virus using thecell's own RNAi machinery that has both high biological activity and invivo stability, and that can effectively inhibit replication of the JCvirus for use in treating pathological processes mediated by JC virusinfection.

SUMMARY OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of the JCvirus in a cell or mammal using such dsRNA. The invention also providescompositions and methods for treating pathological conditions anddiseases caused by JC viral infection, such as PML. The dsRNA of theinvention comprises an RNA strand (the antisense strand) having a regionwhich is less than 30 nucleotides in length, generally 19-24 nucleotidesin length, and is substantially complementary to at least part of anmRNA transcript of a gene from the JC Virus.

In one embodiment, the invention provides double-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression one of the genes ofthe JC virus and viral replication. The dsRNA comprises at least twosequences that are complementary to each other. The dsRNA comprises asense strand comprising a first sequence and an antisense strandcomprising a second sequence. The antisense strand comprises anucleotide sequence which is substantially complementary to at leastpart of an mRNA encoded by a gene from the JC Virus, and the region ofcomplementarity is less than 30 nucleotides in length, generally 19-24nucleotides in length. The dsRNA, upon contacting with a cell expressinginfected with the JC virus, inhibits the expression of a gene from theJC Virus by at least 40%.

For example, the dsRNA molecules of the invention can be comprised of afirst sequence of the dsRNA that is selected from the group consistingof the sense sequences of Tables 1a and b and the second sequence isselected from the group consisting of the antisense sequences of Tables1a and b. The dsRNA molecules of the invention can be comprised ofnaturally occurring nucleotides or can be comprised of at least onemodified nucleotide, such as a 2′-O-methyl modified nucleotide, anucleotide comprising a 5′-phosphorothioate group, and a terminalnucleotide linked to a cholesteryl derivative. Alternatively, themodified nucleotide may be chosen from the group of: a2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide,a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide,2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate,and a non-natural base comprising nucleotide. Generally, such modifiedsequence will be based on a first sequence of said dsRNA selected fromthe group consisting of the sense sequences of Tables 1a and b and asecond sequence selected from the group consisting of the antisensesequences of Tables 1a and 1b.

In another embodiment, the invention provides a cell comprising one ofthe dsRNAs of the invention. The cell is generally a mammalian cell,such as a human cell.

In another embodiment, the invention provides a pharmaceuticalcomposition for inhibiting the replication of the JC virus in anorganism, generally a human subject, comprising one or more of the dsRNAof the invention and a pharmaceutically acceptable carrier or deliveryvehicle.

In another embodiment, the invention provides a method for inhibitingthe expression of a gene in the JC Virus in a cell, comprising thefollowing steps:

-   -   (a) introducing into the cell a double-stranded ribonucleic acid        (dsRNA), wherein the dsRNA comprises at least two sequences that        are complementary to each other. The dsRNA comprises a sense        strand comprising a first sequence and an antisense strand        comprising a second sequence. The antisense strand comprises a        region of complementarity which is substantially complementary        to at least a part of a mRNA encoded by the JC virus, and        wherein the region of complementarity is less than 30        nucleotides in length, generally 19-24 nucleotides in length,        and wherein the dsRNA, upon contact with a cell infected with        the JC virus, inhibits expression of a gene from the JC Virus by        at least 40%; and    -   (b) maintaining the cell produced in step (a) for a time        sufficient to obtain degradation of the mRNA transcript of a JC        virus gene, thereby inhibiting expression of a gene from the JC        Virus in the cell.

In another embodiment, the invention provides methods for treating,preventing or managing pathological processes mediated by JC virusinfection, e.g. such as PML, comprising administering to a patient inneed of such treatment, prevention or management a therapeutically orprophylactically effective amount of one or more of the dsRNAs of theinvention.

In another embodiment, the invention provides vectors for inhibiting theexpression of a gene of the JC virus in a cell, comprising a regulatorysequence operably linked to a nucleotide sequence that encodes at leastone strand of one of the dsRNA of the invention.

In another embodiment, the invention provides a cell comprising a vectorfor inhibiting the expression of a gene of the JC virus in a cell. Thevector comprises a regulatory sequence operably linked to a nucleotidesequence that encodes at least one strand of one of the dsRNA of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

No Figures are presented.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of a gene fromthe JC Virus in a cell or mammal using the dsRNA. The invention alsoprovides compositions and methods for treating pathological conditionsand diseases in a mammal caused by JC virus infection using dsRNA. dsRNAdirects the sequence-specific degradation of mRNA through a processknown as RNA interference (RNAi).

The dsRNA of the invention comprises an RNA strand (the antisensestrand) having a region which is less than 30 nucleotides in length,generally 19-24 nucleotides in length, and is substantiallycomplementary to at least part of an mRNA transcript of a gene from theJC Virus. The use of these dsRNAs enables the targeted degradation ofmRNAs of genes that are implicated in replication and or maintenance ofJC virus infection and the occurrence of PML in a subject infected withthe JC virus. Using cell-based and animal assays, the present inventorshave demonstrated that very low dosages of these dsRNA can specificallyand efficiently mediate RNAi, resulting in significant inhibition ofexpression of a gene from the JC Virus. Thus, the methods andcompositions of the invention comprising these dsRNAs are useful fortreating pathological processes mediated by JC viral infection, e.g.cancer, by targeting a gene involved in JC virus relication and/ormaintenance in a cell.

The following detailed description discloses how to make and use thedsRNA and compositions containing dsRNA to inhibit the expression of agene from the JC virus, as well as compositions and methods for treatingdiseases and disorders caused by the infection with the JC virus, suchas PML. The pharmaceutical compositions of the invention comprise adsRNA having an antisense strand comprising a region of complementaritywhich is less than 30 nucleotides in length, generally 19-24 nucleotidesin length, and is substantially complementary to at least part of an RNAtranscript of a gene from the JC Virus, together with a pharmaceuticallyacceptable carrier.

Accordingly, certain aspects of the invention provide pharmaceuticalcompositions comprising the dsRNA of the invention together with apharmaceutically acceptable carrier, methods of using the compositionsto inhibit expression of a gene in a gene from the JC Virus, and methodsof using the pharmaceutical compositions to treat diseases caused byinfection with the JC virus.

I. Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.However, it will be understood that the term “ribonucleotide” or“nucleotide” can also refer to a modified nucleotide, as furtherdetailed below, or a surrogate replacement moiety. The skilled person iswell aware that guanine, cytosine, adenine, and uracil may be replacedby other moieties without substantially altering the base pairingproperties of an oligonucleotide comprising a nucleotide bearing suchreplacement moiety. For example, without limitation, a nucleotidecomprising inosine as its base may base pair with nucleotides containingadenine, cytosine, or uracil. Hence, nucleotides containing uracil,guanine, or adenine may be replaced in the nucleotide sequences of theinvention by a nucleotide containing, for example, inosine. Sequencescomprising such replacement moieties are embodiments of the invention.

As used herein, “JC virus” refers to the latent polyomavirus JC Virusthat has a reference sequence NC_(—)001699. In addition, furtheraccession numbers of various JC Virus sequences areAB038249.1-AB038255.1, AB048545.1-AB048582.1, AB074575.1-AB074591.1,AB077855.1-AB077879.1, AB081005.1-AB081030.1, AB081600.1-AB081618.1,AB081654.1, AB092578.1-AB092587.1, AB103387.1, AB103402.1-AB103423.1,AB104487.1, AB113118.1-AB113145.1, AB118651.1-AB118659.1,AB126981.1-AB127027.1, AB127342.1, AB127344.1, AB127346.1-AB127349.1,AB127352.1-AB127353.1, AB198940.1-AB198954.1, AB220939.1-AB220943.1,AF004349.1-AF004350.1, AF015526.1-AF015537.1, AF015684.1, AF030085.1,AF281599.1-AF281626.1, AF295731.1-AF295739.1, AF300945.1-AF300967.1,AF363830.1-AF363834.1, AF396422.1-AF396435.1, AY121907.1-AY121915.1,NC_(—)001699.1, U61771.1, U73500.1-U73502.1.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a gene from the JC Virus, including mRNA that is a product of RNAprocessing of a primary transcription product.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butgenerally not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA comprising one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes of the invention.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.

The terms “complementary”, “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide which is “substantially complementaryto at least part of” a messenger RNA (mRNA) refers to a polynucleotidewhich is substantially complementary to a contiguous portion of the mRNAof interest (e.g., encoding JC virus). For example, a polynucleotide iscomplementary to at least a part of a JC virus mRNA if the sequence issubstantially complementary to a non-interrupted portion of a mRNAencoding JC virus.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary, as definedabove, nucleic acid strands. The two strands forming the duplexstructure may be different portions of one larger RNA molecule, or theymay be separate RNA molecules. Where the two strands are part of onelarger molecule, and therefore are connected by an uninterrupted chainof nucleotides between the 3′-end of one strand and the 5′ end of therespective other strand forming the duplex structure, the connecting RNAchain is referred to as a “hairpin loop”. Where the two strands areconnected covalently by means other than an uninterrupted chain ofnucleotides between the 3′-end of one strand and the 5′ end of therespective other strand forming the duplex structure, the connectingstructure is referred to as a “linker”. The RNA strands may have thesame or a different number of nucleotides. The maximum number of basepairs is the number of nucleotides in the shortest strand of the dsRNAminus any overhangs that are present in the duplex. In addition to theduplex structure, a dsRNA may comprise one or more nucleotide overhangs.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches are most tolerated in the terminal regions and,if present, are generally in a terminal region or regions, e.g., within6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

“Introducing into a cell”, when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell”, wherein the cell is part ofa living organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vitro introduction into a cell includes methods known in the art suchas electroporation and lipofection.

The terms “silence” and “inhibit the expression of”, in as far as theyrefer to a gene from the JC Virus, herein refer to the at least partialsuppression of the expression of a gene from the JC Virus, as manifestedby a reduction of the amount of mRNA transcribed from a gene from the JCVirus which may be isolated from a first cell or group of cells in whicha gene from the JC Virus is transcribed and which has or have beentreated such that the expression of a gene from the JC Virus isinhibited, as compared to a second cell or group of cells substantiallyidentical to the first cell or group of cells but which has or have notbeen so treated (control cells). The degree of inhibition is usuallyexpressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to JC virus genometranscription, e.g. the amount of protein encoded by a gene from the JCVirus, or the number of cells displaying a certain phenotype, e.ginfection with the JC Virus. In principle, JC virus genome silencing maybe determined in any cell expressing the target, either constitutivelyor by genomic engineering, and by any appropriate assay. However, when areference is needed in order to determine whether a given dsRNA inhibitsthe expression of a gene from the JC Virus by a certain degree andtherefore is encompassed by the instant invention, the assay provided inthe Examples below shall serve as such reference.

For example, in certain instances, expression of a gene from the JCVirus is suppressed by at least about 20%, 25%, 35%, or 50% byadministration of the double-stranded oligonucleotide of the invention.In some embodiment, a gene from the JC Virus is suppressed by at leastabout 60%, 70%, or 80% by administration of the double-strandedoligonucleotide of the invention. In some embodiments, a gene from theJC Virus is suppressed by at least about 85%, 90%, or 95% byadministration of the double-stranded oligonucleotide of the invention.

As used herein in the context of JC virus expression, the terms “treat”,“treatment”, and the like, refer to relief from or alleviation ofpathological processes mediated by JC virus infection. In the context ofthe present invention insofar as it relates to any of the otherconditions recited herein below (other than pathological processesmediated by JC virus expression), the terms “treat”, “treatment”, andthe like mean to relieve or alleviate at least one symptom associatedwith such condition, or to slow or reverse the progression of suchcondition.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management ofpathological processes mediated by JC virus infection or an overtsymptom of pathological processes mediated by JC virus expression. Thespecific amount that is therapeutically effective can be readilydetermined by ordinary medical practitioner, and may vary depending onfactors known in the art, such as, e.g. the type of pathologicalprocesses mediated by JC virus infection, the patient's history and age,the stage of pathological processes mediated by JC virus infection, andthe administration of other anti-pathological processes mediated by JCvirus infection.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

II. Double-Stranded Ribonucleic Acid (dsRNA)

In one embodiment, the invention provides double-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of a gene from theJC Virus in a cell or mammal, wherein the dsRNA comprises an antisensestrand comprising a region of complementarity which is complementary toat least a part of an mRNA formed in the expression of a gene from theJC Virus, and wherein the region of complementarity is less than 30nucleotides in length, generally 19-24 nucleotides in length, andwherein said dsRNA, upon contact with a cell expressing the gene fromthe JC virus, inhibits the expression of the JC virus gene by at least40%.

The dsRNA comprises two RNA strands that are sufficiently complementaryto hybridize to form a duplex structure. One strand of the dsRNA (theantisense strand) comprises a region of complementarity that issubstantially complementary, and generally fully complementary, to atarget sequence, derived from the sequence of an mRNA formed during theexpression of a gene from the JC Virus, the other strand (the sensestrand) comprises a region which is complementary to the antisensestrand, such that the two strands hybridize and form a duplex structurewhen combined under suitable conditions. Generally, the duplex structureis between 15 and 30, more generally between 18 and 25, yet moregenerally between 19 and 24, and most generally between 19 and 21 basepairs in length. Similarly, the region of complementarity to the targetsequence is between 15 and 30, more generally between 18 and 25, yetmore generally between 19 and 24, and most generally between 19 and 21nucleotides in length. The dsRNA of the invention may further compriseone or more single-stranded nucleotide overhang(s).

The dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc. In a preferred embodiment, a gene from the JC Virus isthe human JC virus genome. In specific embodiments, the antisense strandof the dsRNA comprises the sense sequences of Tables 1a and b and thesecond sequence is selected from the group consisting of the antisensesequences of Tables 1a and b. Alternative antisense agents that targetelsewhere in the target sequence provided in Tables 1a and b can readilybe determined using the target sequence and the flanking JC virussequence.

In further embodiments, the dsRNA comprises at least one nucleotidesequence selected from the groups of sequences provided in Tables 1a andb. In other embodiments, the dsRNA comprises at least two sequencesselected from this group, wherein one of the at least two sequences iscomplementary to another of the at least two sequences, and one of theat least two sequences is substantially complementary to a sequence ofan mRNA generated in the expression of a gene from the JC Virus.Generally, the dsRNA comprises two oligonucleotides, wherein oneoligonucleotide is described as the sense strand in Tables 1a and b andthe second oligonucleotide is described as the antisense strand inTables 1a and b

The skilled person is well aware that dsRNAs comprising a duplexstructure of between 20 and 23, but specifically 21, base pairs havebeen hailed as particularly effective in inducing RNA interference(Elbashir et al., EMBO 2001, 20:6877-6888). However, others have foundthat shorter or longer dsRNAs can be effective as well. In theembodiments described above, by virtue of the nature of theoligonucleotide sequences provided in Tables 1a and b, the dsRNAs of theinvention can comprise at least one strand of a length of minimally 21nt. It can be reasonably expected that shorter dsRNAs comprising one ofthe sequences of Tables 1a and b minus only a few nucleotides on one orboth ends may be similarly effective as compared to the dsRNAs describedabove. Hence, dsRNAs comprising a partial sequence of at least 15, 16,17, 18, 19, 20, or more contiguous nucleotides from one of the sequencesof Tables 1a and b, and differing in their ability to inhibit theexpression of a gene from the JC Virus in a FACS assay as describedherein below by not more than 5, 10, 15, 20, 25, or 30% inhibition froma dsRNA comprising the full sequence, are contemplated by the invention.Further dsRNAs that cleave within the target sequence provided in Tables1a and b can readily be made using the JC virus sequence and the targetsequence provided.

In addition, the RNAi agents provided in Tables 1a and b identify a sitein the JC virus mRNA that is susceptible to RNAi based cleavage. As suchthe present invention further includes RNAi agents that target withinthe sequence targeted by one of the agents of the present invention. Asused herein a second RNAi agent is said to target within the sequence ofa first RNAi agent if the second RNAi agent cleaves the message anywherewithin the mRNA that is complementary to the antisense strand of thefirst RNAi agent. Such a second agent will generally consist of at least15 contiguous nucleotides from one of the sequences provided in Tables1a and b coupled to additional nucleotide sequences taken from theregion contiguous to the selected sequence in a gene from the JC Virus.For example, the last 15 nucleotides of SEQ ID NO:1 combined with thenext 6 nucleotides from the target JC virus genome produces a singlestrand agent of 21 nucleotides that is based on one of the sequencesprovided in Tables 1a and b.

The dsRNA of the invention can contain one or more mismatches to thetarget sequence. In a preferred embodiment, the dsRNA of the inventioncontains no more than 3 mismatches. If the antisense strand of the dsRNAcontains mismatches to a target sequence, it is preferable that the areaof mismatch not be located in the center of the region ofcomplementarity. If the antisense strand of the dsRNA containsmismatches to the target sequence, it is preferable that the mismatch berestricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or1 nucleotide from either the 5′ or 3′ end of the region ofcomplementarity. For example, for a 23 nucleotide dsRNA strand which iscomplementary to a region of a gene from the JC Virus, the dsRNAgenerally does not contain any mismatch within the central 13nucleotides. The methods described within the invention can be used todetermine whether a dsRNA containing a mismatch to a target sequence iseffective in inhibiting the expression of a gene from the JC Virus.Consideration of the efficacy of dsRNAs with mismatches in inhibitingexpression of a gene from the JC Virus is important, especially if theparticular region of complementarity in a gene from the JC Virus isknown to have polymorphic sequence variation within the population.

In one embodiment, at least one end of the dsRNA has a single-strandednucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAshaving at least one nucleotide overhang have unexpectedly superiorinhibitory properties than their blunt-ended counterparts. Moreover, thepresent inventors have discovered that the presence of only onenucleotide overhang strengthens the interference activity of the dsRNA,without affecting its overall stability. dsRNA having only one overhanghas proven particularly stable and effective in vivo, as well as in avariety of cells, cell culture mediums, blood, and serum. Generally, thesingle-stranded overhang is located at the 3′-terminal end of theantisense strand or, alternatively, at the 3′-terminal end of the sensestrand. The dsRNA may also have a blunt end, generally located at the5′-end of the antisense strand. Such dsRNAs have improved stability andinhibitory activity, thus allowing administration at low dosages, i.e.,less than 5 mg/kg body weight of the recipient per day. Generally, theantisense strand of the dsRNA has a nucleotide overhang at the 3′-end,and the 5′-end is blunt. In another embodiment, one or more of thenucleotides in the overhang is replaced with a nucleoside thiophosphate.

In yet another embodiment, the dsRNA is chemically modified to enhancestability. The nucleic acids of the invention may be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Specific examples of preferred dsRNAcompounds useful in this invention include dsRNAs containing modifiedbackbones or no natural internucleoside linkages. As defined in thisspecification, dsRNAs having modified backbones include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of this specification,and as sometimes referenced in the art, modified dsRNAs that do not havea phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified dsRNA backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference

Preferred modified dsRNA backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatoms and alkyl orcycloalkyl internucleoside linkages, or ore or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other preferred dsRNA mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an dsRNA mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar backbone of an dsRNA is replacedwith an amide containing backbone, in particular an aminoethylglycinebackbone. The nucleobases are retained and are bound directly orindirectly to aza nitrogen atoms of the amide portion of the backbone.Representative U.S. patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found in Nielsen et al.,Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are dsRNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH.sub.2-NH—CH.sub.2-,—CH.sub.2-N(CH.sub.3)-O—CH.sub.2-[known as a methylene (methylimino) orMMI backbone], —CH.sub.2-O—N(CH.sub.3)-CH.sub.2-,—CH.sub.2-N(CH.sub.3)-N(CH.sub.3)-CH.sub.2- and—N(CH.sub.3)-CH.sub.2-CH.sub.2-[wherein the native phosphodiesterbackbone is represented as —O—P—O—CH.sub.2-] of the above-referencedU.S. Pat. No. 5,489,677, and the amide backbones of the above-referencedU.S. Pat. No. 5,602,240. Also preferred are dsRNAs having morpholinobackbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified dsRNAs may also contain one or more substituted sugar moieties.Preferred dsRNAs comprise one of the following at the 2′ position: OH;F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C.sub.1 to C.sub.10 alkyl or C.sub.2 toC.sub.10 alkenyl and alkynyl. Particularly preferred areO[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,O(CH.sub.2).sub.nONH.sub.2, andO(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.su-b.3)].sub.2, where n and m arefrom 1 to about 10. Other preferred dsRNAs comprise one of the followingat the 2′ position: C.sub.1 to C.sub.10 lower alkyl, substituted loweralkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl,Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2,NO.sub.2, N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an dsRNA, or a group for improving thepharmacodynamic properties of an dsRNA, and other substituents havingsimilar properties. A preferred modification includes 2′-methoxyethoxy(2′-O—CH.sub.2CH.sub.2OCH.sub.3, also known as 2′-O-(2-methoxyethyl) or2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., analkoxy-alkoxy group. A further preferred modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH.sub.2).sub.2ON(CH.sub.3).sub.2group, also known as 2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH.sub.2-O—CH.sub.2-N(CH.sub.2).sub.2, also described in exampleshereinbelow.

Other preferred modifications include 2′-methoxy (2′-OCH.sub.3),2′-aminopropoxy (2′-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2′-fluoro(2′-F). Similar modifications may also be made at other positions on thedsRNA, particularly the 3′ position of the sugar on the 3′ terminalnucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminalnucleotide. DsRNAs may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative U.S.patents that teach the preparation of such modified sugar structuresinclude, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800;5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

dsRNAs may also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T.and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

Another modification of the dsRNAs of the invention involves chemicallylinking to the dsRNA one or more moieties or conjugates which enhancethe activity, cellular distribution or cellular uptake of the dsRNA.Such moieties include but are not limited to lipid moieties such as acholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 199,86, 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let.,1994 4 1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan etal., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Biorg.Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser etal., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

Representative U.S. patents that teach the preparation of such dsRNAconjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979;4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538;5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporatedby reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an dsRNA. The present invention also includesdsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compoundsor “chimeras,” in the context of this invention, are dsRNA compounds,particularly dsRNAs, which contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of an dsRNA compound. These dsRNAs typically contain atleast one region wherein the dsRNA is modified so as to confer upon thedsRNA increased resistance to nuclease degradation, increased cellularuptake, and/or increased binding affinity for the target nucleic acid.An additional region of the dsRNA may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency of dsRNA inhibition ofgene expression. Consequently, comparable results can often be obtainedwith shorter dsRNAs when chimeric dsRNAs are used, compared tophosphorothioate deoxydsRNAs hybridizing to the same target region.Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the dsRNA may be modified by a non-ligand group. Anumber of non-ligand molecules have been conjugated to dsRNAs in orderto enhance the activity, cellular distribution or cellular uptake of thedsRNA, and procedures for performing such conjugations are available inthe scientific literature. Such non-ligand moieties have included lipidmoieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci.USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharanet al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg.Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiolor undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111;Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie,1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923). Representative United States patents thatteach the preparation of such dsRNA conjugates have been listed above.Typical conjugation protocols involve the synthesis of dsRNAs bearing anaminolinker at one or more positions of the sequence. The amino group isthen reacted with the molecule being conjugated using appropriatecoupling or activating reagents. The conjugation reaction may beperformed either with the dsRNA still bound to the solid support orfollowing cleavage of the dsRNA in solution phase. Purification of thedsRNA conjugate by HPLC typically affords the pure conjugate.

Vector Encoded RNAi Agents

The dsRNA of the invention can also be expressed from recombinant viralvectors intracellularly in vivo. The recombinant viral vectors of theinvention comprise sequences encoding the dsRNA of the invention and anysuitable promoter for expressing the dsRNA sequences. Suitable promotersinclude, for example, the U6 or H1 RNA pol III promoter sequences andthe cytomegalovirus promoter. Selection of other suitable promoters iswithin the skill in the art. The recombinant viral vectors of theinvention can also comprise inducible or regulatable promoters forexpression of the dsRNA in a particular tissue or in a particularintracellular environment. The use of recombinant viral vectors todeliver dsRNA of the invention to cells in vivo is discussed in moredetail below.

dsRNA of the invention can be expressed from a recombinant viral vectoreither as two separate, complementary RNA molecules, or as a single RNAmolecule with two complementary regions.

Any viral vector capable of accepting the coding sequences for the dsRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of viral vectors can be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses, or by substituting different viral capsidproteins, as appropriate.

For example, lentiviral vectors of the invention can be pseudotyped withsurface proteins from vesicular stomatitis virus (VSV), rabies, Ebola,Mokola, and the like. AAV vectors of the invention can be made to targetdifferent cells by engineering the vectors to express different capsidprotein serotypes. For example, an AAV vector expressing a serotype 2capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsidgene in the AAV 2/2 vector can be replaced by a serotype 5 capsid geneto produce an AAV 2/5 vector. Techniques for constructing AAV vectorswhich express different capsid protein serotypes are within the skill inthe art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801,the entire disclosure of which is herein incorporated by reference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe dsRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat.Genet. 33: 401-406, the entire disclosures of which are hereinincorporated by reference.

Preferred viral vectors are those derived from AV and AAV. In aparticularly preferred embodiment, the dsRNA of the invention isexpressed as two separate, complementary single-stranded RNA moleculesfrom a recombinant AAV vector comprising, for example, either the U6 orH1 RNA promoters, or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA of the invention, a methodfor constructing the recombinant AV vector, and a method for deliveringthe vector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA of the invention, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski Ret al. (1987),J. Virol. 61: 3096-3101; Fisher K Jet al. (1996), J. Virol, 70: 520-532;Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No.5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No.WO 94/13788; and International Patent Application No. WO 93/24641, theentire disclosures of which are herein incorporated by reference.

III. Pharmaceutical Compositions Comprising dsRNA

In one embodiment, the invention provides pharmaceutical compositionscomprising a dsRNA, as described herein, and a pharmaceuticallyacceptable carrier. The pharmaceutical composition comprising the dsRNAis useful for treating a disease or disorder associated with theexpression or activity of a gene from the JC Virus and/or viralinfection, such as PML. Such pharmaceutical compositions are formulatedbased on the mode of delivery. One example is compositions that areformulated for systemic administration via parenteral delivery.

The pharmaceutical compositions of the invention are administered indosages sufficient to inhibit expression of a gene from the JC Virus.The present inventors have found that, because of their improvedefficiency, compositions comprising the dsRNA of the invention can beadministered at surprisingly low dosages. A maximum dosage of 5 mg dsRNAper kilogram body weight of recipient per day is sufficient to inhibitor completely suppress expression of a gene from the JC Virus.

In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0milligrams per kilogram body weight of the recipient per day, generallyin the range of 1 microgram to 1 mg per kilogram body weight per day.The pharmaceutical composition may be administered once daily, or thedsRNA may be administered as two, three, or more sub-doses atappropriate intervals throughout the day or even using continuousinfusion or delivery through a controlled release formulation. In thatcase, the dsRNA contained in each sub-dose must be correspondinglysmaller in order to achieve the total daily dosage. The dosage unit canalso be compounded for delivery over several days, e.g., using aconventional sustained release formulation which provides sustainedrelease of the dsRNA over a several day period. In this embodiment, thedosage unit contains a corresponding multiple of the daily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pathological processesmediated by JC virus expression. Such models are used for in vivotesting of dsRNA, as well as for determining a therapeutically effectivedose.

The present invention also includes pharmaceutical compositions andformulations which include the dsRNA compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical, pulmonary, e.g., by inhalation or insufflation of powders oraerosols, including by nebulizer; intratracheal, intranasal, epidermaland transdermal), oral or parenteral. Parenteral administration includesintravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; or intracranial, e.g., intrathecalor intraventricular, administration.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G.sub.M1, or (B)is derivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G.sub.M1,galactocerebroside sulfate and phosphatidylinositol to improve bloodhalf-lives of liposomes. These findings were expounded upon by Gabizonet al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No.4,837,028 and WO 88/04924, both to Allen et al., disclose liposomescomprising (1) sphingomyelin and (2) the ganglioside G.sub.M1 or agalactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.)discloses liposomes comprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Limet al).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C.sub.1215G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in theart. WO 96/40062 to Thierry et al. discloses methods for encapsulatinghigh molecular weight nucleic acids in liposomes. U.S. Pat. No.5,264,221 to Tagawa et al. discloses protein-bonded liposomes andasserts that the contents of such liposomes may include an dsRNA RNA.U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods ofencapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love etal. discloses liposomes comprising dsRNA dsRNAs targeted to the rafgene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Agents that enhance uptake of dsRNAs at the cellular level may also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antisense compounds and (b) one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited todaunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosinearabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphor-amide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulation a range of dosage for use in humans. The dosage ofcompositions of the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the compound or, when appropriate, of thepolypeptide product of a target sequence (e.g., achieving a decreasedconcentration of the polypeptide) that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In addition to their administration individually or as a plurality, asdiscussed above, the dsRNAs of the invention can be administered incombination with other known agents effective in treatment ofpathological processes mediated by JC virus expression. In any event,the administering physician can adjust the amount and timing of dsRNAadministration on the basis of results observed using standard measuresof efficacy known in the art or described herein.

Methods for Treating Diseases Caused by Expression of a Gene from the JCVirus

The invention relates in particular to the use of a dsRNA or apharmaceutical composition prepared therefrom for the treatment orprevention of pathological conditions associated with JC Virusinfection, e.g., PML. Owing to the inhibitory effect on JC virusexpression, an dsRNA according to the invention or a pharmaceuticalcomposition prepared therefrom can enhance the quality of life,particularly in a patient being treated with an anti-VLA4 antibody aspart of treatment for MS.

The invention furthermore relates to the use of an dsRNA or apharmaceutical composition thereof for treating PML in combination withother pharmaceuticals and/or other therapeutic methods, e.g., with knownpharmaceuticals and/or known therapeutic methods, such as, for example,those which are currently employed for treating cancer and/or forpreventing tumor metastasis. Preference is given to a combination withradiation therapy and chemotherapeutic agents, such as cisplatin,cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen.

The invention can also be practiced by including with a specific RNAiagent, in combination with another anti-cancer chemotherapeutic agent,such as any conventional chemotherapeutic agent. The combination of aspecific binding agent with such other agents can potentiate thechemotherapeutic protocol. Numerous chemotherapeutic protocols willpresent themselves in the mind of the skilled practitioner as beingcapable of incorporation into the method of the invention. Anychemotherapeutic agent can be used, including alkylating agents,antimetabolites, hormones and antagonists, radioisotopes, as well asnatural products. For example, the compound of the invention can beadministered with antibiotics such as doxorubicin and otheranthracycline analogs, nitrogen mustards such as cyclophosphamide,pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, taxoland its natural and synthetic derivatives, and the like. As anotherexample, in the case of mixed tumors, such as adenocarcinoma of thebreast, where the tumors include gonadotropin-dependent andgonadotropin-independent cells, the compound can be administered inconjunction with leuprolide or goserelin (synthetic peptide analogs ofLH-RH). Other antineoplastic protocols include the use of a tetracyclinecompound with another treatment modality, e.g., surgery, radiation,etc., also referred to herein as “adjunct antineoplastic modalities.”Thus, the method of the invention can be employed with such conventionalregimens with the benefit of reducing side effects and enhancingefficacy.

Methods for Inhibiting Expression of a Gene from the JC Virus

In yet another aspect, the invention provides a method for inhibitingthe expression of a gene from the JC Virus in a mammal. The methodcomprises administering a composition of the invention to the mammalsuch that expression of the target JC virus genome is silenced. Becauseof their high specificity, the dsRNAs of the invention specificallytarget RNAs (primary or processed) of the target JC virus gene.Compositions and methods for inhibiting the expression of these JC virusgenes using dsRNAs can be performed as described elsewhere herein.

In one embodiment, the method comprises administering a compositioncomprising a dsRNA, wherein the dsRNA comprises a nucleotide sequencewhich is complementary to at least a part of an RNA transcript of a genefrom the JC Virus, to the mammal to be treated. When the organism to betreated is a mammal such as a human, the composition may be administeredby any means known in the art including, but not limited to oral orparenteral routes, including intravenous, intramuscular, subcutaneous,transdermal, airway (aerosol), nasal, administration. In preferredembodiments, the compositions are administered by intravenous infusionor injection.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

Examples

Design of JCV siRNAs

Full-length genome sequences to JC virus available on Apr. 10, 2006,were obtained, resulting in a target pool of 388 sequences (accessionnumbers: AB038249.1-AB038255.1; AB048545.1-AB048582.1;AB074575.1-AB074591.1; AB077855.1-AB077879.1; AB081005.1-AB081030.1;AB081600.1-AB081618.1; AB081654.1; AB092578.1-AB092587.1; AB103387.1;AB103402.1-AB103423.1; AB104487.1; AB113118.1-AB113145.1;AB118651.1-AB118659.1; AB126981.1-AB127027.1; AB127342.1; AB127344.1;AB127346.1-AB127349.1; AB127352.1-AB127353.1; AB198940.1-AB198954.1;AB220939.1-AB220943.1; AF004349.1-AF004350.1; AF015526.1-AF015537.1;AF015684.1; AF030085.1; AF281599.1-AF281626.1; AF295731.1-AF295739.1;AF300945.1-AF300967.1; AF363830.1-AF363834.1; AF396422.1-AF396435.1;AY121907.1-AY121915.1; NC_(—)001699.1; U61771.1; U73500.1-U73502.1).NC_(—)001699 was defined as reference sequence.

The siRNA selection process was run as follows: ClustalW multiplealignment was used to generate a global alignment of all sequences fromthe target pool. An IUPAC consensus sequence was then generated.

All conserved 19mer target sequences from the IUPAC consensusrepresented by stretches containing only A, T, C or G bases, which aretherefore present in all sequences of the target pool were selected. Inorder to only select siRNAs that target transcribed sequence parts ofthe JC virus, candidate target sequences were selected out of the poolof conserved 19mer target sequences. For this, candidate targetsequences covering regions between nucleotide 163-2594 and between2527-5115 relative to reference sequence were extracted for late andearly genes, respectively. Further, as sequences for early genes are inreverse complement orientation compared with genomic sequences,candidate target sequences of these genes were transferred to reversecomplement sequences and replaced the former pool of candidate targetsequences.

In order to rank candidate target sequences and their respective siRNAsand select appropriate ones, their predicted potential for interactingwith irrelevant targets (off-target potential) was taken as a rankingparameter. siRNAs with low off-target potential were defined aspreferable and assumed to be more specific in vivo.

For predicting siRNA-specific off-target potential, the followingassumptions were made:

-   -   1) positions 2 to 9 (counting 5′ to 3′) of a strand (seed        region) may contribute more to off-target potential than rest of        sequence (non-seed and cleavage site region)    -   2) positions 10 and 11 (counting 5′ to 3′) of a strand (cleavage        site region) may contribute more to off-target potential than        non-seed region    -   3) an off-target score can be calculated for each hit, based on        identity to siRNA sequence and position of mismatches    -   4) assuming potential abortion of sense strand activity by        internal modifications introduced, only off-target potential of        antisense strand will be relevant

To identify potential off-target genes, 19mer input sequences weresubjected to a homology search against publically available human mRNAsequences.

To this purpose, fastA (version 3.4) searches were performed with all19mer candidate target sequences against a human RefSeq database(downloaded available version from ftp://ftp.ncbi.nih.gov/refseq/ onNov. 7, 2006). FastA searches were executed withparameters-values-pairs—f 50-g 50 in order to take into account thehomology over the full length of the 19mer without any gaps. In order toensure the listing of all relevant off-target hits in the fastA outputfile the parameter—E 30000 was used in addition. A scoring matrix wasapplied for the run that assessed every nucleotide match with a score of13 and every mismatch with a score of −7. The search resulted in a listof potential off-targets for each candidate siRNA.

To sort the resulting list of potential off-targets for each siRNA,fastA output files were analyzed to identify the best off-target and itsoff-target score. The following off-target properties for each 19merinput sequence were extracted for each off-target to calculate theoff-target score:

-   -   Number of mismatches in non-seed region    -   Number of mismatches in seed region    -   Number of mismatches in cleavage site region

The off-target score was calculated for considering assumption 1 to 3 asfollows:

Off-target  score = number  of  seed  mismatches * 10 + number  of  cleavage  site  mismatches * 1.2 + number  of  non-seed  mismatches * 1

The most relevant off-target gene for input each 19mer input sequenceswas defined as the gene with the lowest off-target score. Accordingly,the lowest off-target score was defined as the relevant off-target scorefor the corresponding siRNA.

In order to generate a ranking for siRNAs, calculated relevantoff-target scores were transferred into a result table. All siRNAs weresorted according to the off-target score (descending).

An off-target score of 2.2 was defined as cut-off for siRNA selection(specificity criterion). In addition, all sequences with only onemismatch in the seed region were eliminated from the screening set. Theselection procedure resulted in a set of 93 JCV specific siRNAs (Table1a).

An expanded screening was generated by re-calculating the predictedspecificity based on the newly available human RefSeq database (HumanmRNA sequences in RefSeq release version 21 (downloaded Jan. 12, 2007))and selecting only 208 siRNAs that did not contain more than 3 G's in arow and had an off-target score of at least 2 for the antisense strand(Table 1b).

Synthesis of JCV siRNAs

All siRNAs were synthesized in 0.2 μmole synthesis scale on an ABI3900DNA synthesizer according to standard procedures.

For the initial screening set (93 different siRNA sequences), 4different strategies of chemical modification were used:

a) exo/endo light (EEL):

-   -   sense strand: 2′-O-methyl @ all pyrimidines, PTO between        nucleotides 20 and 21 (counting from 5′-end), dTdT at 3′-end        (nucleotides 20 and 21)    -   antisense strand: 2′-O-methyl at pyrimidines in 5′-UA-3′ and        5′-CA-3′ motives, PTO between nucleotides 20 and 21 (counting        from 5′-end), dTdT at 3′-end (nucleotides 20 and 21)

b) EEL plus 2′-O-methyl in position 2 of antisense strand (only if no5′-UA-3′ and 5′-CA-3′ at 5′-end, otherwise already covered by EEL)

c) EEL plus 2′-O-methyl in position 2 of sense strand (only if nopyrimidine in position 2, otherwise already covered by EEL)

d) EEL plus 2′-O-methyl in position 2 of sense and antisense strand(only if not already covered by a, b, and c) (Table 1a)

For the expanded screening set (208 different siRNA sequences), siRNAswere composed of unmodified RNA oligonucleotides with dT/dT overhangs(dTdT at 3′-end (nucleotides 20 and 21) of antisense and sense strands)(Table 1b).

Screening of JCV siRNAs

Construction of Reporter-Systems Encoding JCV Transcripts

The sequence of the early JCV transcript (E) was synthesized at GENEART(Regensburg, Germany) and cloned into GENEART standard vectors. Thesequence of the late JCV transcript was subdivided in a first approachinto two fragments: L1, including the transcript sequence of the VP1protein, and LA23, including the sequences of VP2, VP3 and theAgnoprotein. Due to cloning problems with fragment LA23, this sequencewas subdivided in a second approach into two fragments (LA23 1-700 andLA23 701-1438). All sequences were synthesized at GENEART and clonedinto GENEART standard vectors. All fragments (E, L1, LA23 1-700 and LA23701-1438) were subcloned into psiCheck-2 (Promega, Mannheim, Germany)via XhoI and NotI (both NEB, Frankfurt, Germany), resulting inconstructs with the JCV sequences between the stop-codon and thepolyA-signal of Renilla luciferase.

L1 (SEQ ID NO: 931) CTCGAGACTTTTAGGGTTGTACGGGACTGTAACACCTGCTCTTGAAGCATATGAAGATGGCCCCAACAAAAAGAAAAGGAGAAAGGAAGGACCCCGTGCAAGTTCCAAAACTTCTTATAAGAGGAGGAGTAGAAGTTCTAGAAGTTAAAACTGGGGTTGACTCAATTACAGAGGTAGAATGCTTTTTAACTCCAGAAATGGGTGACCCAGATGAGCATCTTAGGGGTTTTAGTAAGTCAATTTCTATATCAGATACATTTGAAAGTGACTCCCCAAATAAGGACATGCTTCCTTGTTACAGTGTGGCCAGAATTCCACTACCCAATCTAAATGAGGATCTAACCTGTGGAAATATACTAATGTGGGAGGCTGTGACCTTAAAAACTGAGGTTCTAGGGGTGACAACTTTGATGAATGTGCACTCTAATGGTCAAGCAACTCATGACAATGGTGCAGGAAAGCCAGTGCAGGGCACCAGCTTTCATTTTTTTTCTGTTGGCGGGGAGGCTTTAGAATTACAGGGGGTGGTTTTTAATTACAGAACAAAGTACCCAGATGGAACAATTTTTCCAAAGAATGCAACAGTGCAATCTCAAGTAATGAACACAGAGCACAAGGCGTACCTAGATAAGAACAAAGCATATCCTGTTGAATGTTGGGTTCCTGATCCCACCAGAAATGAAAACACAAGATATTTTGGGACACTAACAGGAGGAGAAAATGTTCCTCCAGTTCTTCATATAACAAACACTGCCACAACAGTGCTGCTTGATGAATTTGGTGTTGGGCCACTTTGCAAAGGTGACAACTTGTATTTGTCAGCTGTTGATGTTTGTGGAATGTTTACTAACAGATCTGGTTCCCAGCAGTGGAGAGGACTGTCCAGATATTTTAAGGTTCAGCTCAGAAAAAGGAGGGTTAAAAACCCCTACCCAATTTCTTTCCTTCTTACTGATTTGATTAACAGAAGGACCCCTAGAGTTGATGGGCAACCTATGTATGGTATGGATGCTCAGGTAGAGGAGGTTAGAGTTTTTGAGGGGACAGAGGAACTTCCAGGGGACCCAGACATGATGAGATATGTTGACAGATATGGACAGTTGCAAACAAAGATGCTGTAATCAAAATCCTTTATTGTAATATGCAGTACATTTTAATAAAGTATAACCAGCTTTACTTTACAGTTGCAGTCATGCGGCC GC E (SEQ ID NO: 932)CTCGAGCCGCCTCCAAGCTTACTCAGAAGTAGTAAGGGCGTGGAGGCTTTTTAGGAGGCCAGGGAAATTCCCTTGTTTTTCCCTTTTTTGCAGTAATTTTTTGCTGCAAAAAGCTAAAATGGACAAAGTGCTGAATAGGGAGGAATCCATGGAGCTTATGGATTTATTAGGCCTTGATAGGTCTGCATGGGGGAACATTCCTGTCATGAGAAAAGCTTATCTGAAAAAATGCAAAGAACTCCACCCTGATAAAGGTGGGGACGAAGACAAGATGAAGAGAATGAATTTTTTATATAAAAAAATGGAACAAGGTGTAAAAGTTGCTCATCAGCCTGATTTTGGTACATGGAATAGTTCAGAGGTTGGTTGTGATTTTCCTCCTAATTCTGATACCCTTTATTGCAAGGAATGGCCTAACTGTGCCACTAATCCTTCAGTGCATTGCCCCTGTTTAATGTGCATGCTAAAATTAAGGCATAGAAACAGAAAATTTTTAAGAAGCAGCCCACTTGTGTGGATAGATTGCTATTGCTTTGATTGCTTCAGACAATGGTTTGGGTGTGACTTAACCCAAGAAGCTCTTCATTGCTGGGAGAAAGTTCTTGGAGACACCCCCTACAGGGATCTAAAGCTTTAAGTGCCAACCTATGGAACAGATGAATGGGAATCCTGGTGGAATACATTTAATGAGAAGTGGGATGAAGACCTGTTTTGCCATGAAGAAATGTTTGCCAGTGATGATGAAAACACAGGATCCCAACACTCTACCCCACCTAAAAAGAAAAAAAAGGTAGAAGACCCTAAAGACTTTCCTGTAGATCTGCATGCATTCCTCAGTCAAGCTGTGTTTAGTAATAGAACTGTTGCTTCTTTTGCTGTGTATACCACTAAAGAAAAAGCTCAAATTTTATATAAGAAACTTATGGAAAAATATTCTGTAACTTTTATAAGTAGACATGGTTTTGGGGGTCATAATATTTTGTTTTTCTTAACACCACATAGACATAGAGTGTCAGCAATTAATAACTACTGTCAAAAACTATGTACCTTTAGTTTTTTAATTTGTAAAGGTGTGAATAAGGAATACTTGTTTTATAGTGCCCTGTGTAGACAGCCATATGCAGTAGTGGAAGAAAGTATTCAGGGGGGCCTTAAGGAGCATGACTTTAACCCAGAAGAACCAGAAGAAACTAAGCAGGTTTCATGGAAATTAGTTACACAGTATGCCTTGGAAACCAAGTGTGAGGATGTTTTTTTGCTTATGGGCATGTACTTAGACTTTCAGGAAAACCCACAGCAATGCAAAAAATGTGAAAAAAAGGATCAGCCAAATCACTTTAACCATCATGAAAAACACTATTATAATGCCCAAATTTTTGCAGATAGCAAAAATCAAAAAAGCATTTGCCAGCAGGCTGTTGATACTGTAGCAGCCAAACAAAGGGTTGACAGCATCCACATGACCAGAGAAGAAATGTTAGTTGAAAGGTTTAATTTCTTGCTTGATAAAATGGACTTAATTTTTGGGGCACATGGCAATGCTGTTTTAGAGCAATATATGGCTGGGGTGGCCTGGATTCATTGCTTGCTGCCTCAAATGGACACTGTTATTTATGACTTTCTAAAATGCATTGTATTAAACATTCCAAAAAAAAGGTACTGGCTATTCAAGGGGCCAATAGACAGTGGCAAAACTACTTTAGCTGCAGCTTTACTTGATCTCTGTGGGGGAAAGTCATTAAATGTTAATATGCCATTAGAAAGATTAAACTTTGAATTAGGAGTGGGTATAGATCAGTTTATGGTTGTATTTGAGGATGTAAAAGGCACTGGTGCAGAGTCAAGGGATTTACCTTCAGGGCATGGCATAAGCAACCTTGATTGCTTAAGAGATTACTTAGATGGAAGTGTAAAAGTTAATTTAGAGAGAAAACACCAAAACAAAAGAACACAGGTGTTTCCACCTGGAATTGTAACCATGAATGAATATTCAGTGCCTAGAACTTTACAGGCCAGATTTGTAAGGCAGATAGATTTTAGACCAAAGGCCTACCTGAGAAAATCACTAAGCTGCTCTGAGTATTTGCTAGAAAAAAGGATTTTGCAAAGTGGTATGACTTTGCTTTTGCTTTTAATCTGGTTTAGGCCAGGTTGCTGACTTTGCAGCTGCCATTCATGAGAGGATTGTGCAGTGGAAAGAAAGGCTGGATTTAGAAATAAGCATGTATACATTTTCTACTATGAAAGCTAATGTTGGTATGGGGAGACCCATTCTTGACTTTCCTAGAGAGGAAGATTCTGAAGCAGAAGACTCTGGACATGGATCAAGCACGTAATCACAATCACAATGCTTTTCCCAGGTCTCAGAAGCCTCTGGTGCAGACACACAGGAAAACTGCACTTTTCACATCTGTAAAGGCTTTCAATGTTTCAAAAAACCAAAGACCCCTCCCCCAAAATAACTGCAACTGTGCGGCCGC LA23 1-700 (SEQ ID NO: 933)CTCGAGCAGCTAACAGCCAGTAAACAAAGCACAAGGGGAAGTTGAAAGCAGCCAAGGGAACATGTTTTGCGAGCCAGAGCTGTTTTGGCTTGTCACCAGCTGGCCATGGTTCTTCGCCAGCTGTCACGTAAGGCTTCTGTGAAAGTTAGTAAAACCTGGAGTGGAACTAAAAAAAGAGCTCAAAGGATTTTAATTTTTTTGTTAGAATTTTTGCTGGACTTTTGCACAGGTGAAGACAGTGTAGACGGGAAAAAAAGACAGAGACACAGTGGTTTGACTGAGCAGACATACAGTGCTTTGCCTGAACCAAAAGCTACATAGGTAAGTAATGTTTTTTTTTGTGTTTTCAGGTTCATGGGTGCCGCACTTGCACTTTTGGGGGACCTAGTTGCTACTGTTTCTGAGGCTGCTGCTGCCACAGGATTTTCAGTAGCTGAAATTGCTGCTGGAGAGGCTGCTGCTACTATAGAAGTTGAAATTGCATCCCTTGCTACTGTAGAGGGGATTACAAGTACCTCTGAGGCTATAGCTGCTATAGGCCTTACTCCTGAAACATATGCTGTAATAACTGGAGCTCCGGGGGCTGTAGCTGGGTTTGCTGCATTGGTTCAAACTGTAACTGGTGGTAGTGCTATTGCTCAGTTGGGATATAGATTTTTTGCTGACTGGGATCATAAAGTTTCAACAGTTGGGCTTTTTC GCGGCCGCLA23 701-1438 (SEQ ID NO: 934)CTCGAGAGCAGCCAGCTATGGCTTTACAATTATTTAATCCAGAAGACTACTATGATATTTTATTTCCTGGAGTGAATGCCTTTGTTAACAATATTCACTATTTAGATCCTAGACATTGGGGCCCGTCCTTGTTCTCCACAATCTCCCAGGCTTTTTGGAATCTTGTTAGAGATGATTTGCCAGCCTTAACCTCTCAGGAAATTCAGAGAAGAACCCAAAAACTATTTGTTGAAAGTTTAGCAAGGTTTTTGGAAGAAACTACTTGGGCAATAGTTAATTCACCAGCTAACTTATATAATTATATTTCAGACTATTATTCTAGATTGTCTCCAGTTAGGCCCTCTATGGTAAGGCAAGTTGCCCAAAGGGAGGGAACCTATATTTCTTTTGGCCACTCATACACCCAAAGTATAGATGATGCAGACAGCATTCAAGAAGTTACCCAAAGGCTAGATTTAAAAACCCCAAATGTGCAATCTGGTGAATTTATAGAAAGAAGTATTGCACCAGGAGGTGCAAATCAAAGATCTGCTCCTCAATGGATGTTGCCTTTACTTTTAGGGTTGTACGGGACTGTAACACCTGCTCTTGAAGCATATGAAGATGGCCCCAACAAAAAGAAAAGGAGAAAGGAAGGACCCCGTGCAAGTTCCAAAACTTCTTATAAGAGGAGGAGTAGAAGTTCTAGAAGTTAAAACTGGGGTTGACTCAATTACAGAGGTAGAATGCTGCGGCCGC

Screen of JCV siRNAs in Transfected Cells

Cos-7 cells (DSMZ, Braunschweig, Germany, #ACC-60) were seeded at1.5×10⁴ cells/well on white 96-well plates with clear bottoms (GreinerBio-One GmbH, Frickenhausen, Germany) in 75 μl of growth medium.Directly after seeding the cells, 50 ng of the correspondingreporter-plasmid per well was transfected with Lipofectamine™ 2000(Invitrogen GmbH, Karlsruhe, Germany), with the plasmid diluted inOpti-MEM to a final volume of 12.5 μl per well, prepared as a mastermixfor the whole plate.

4 h after plasmid transfection, growth medium was removed from cells andreplaced by 100 μl/well of fresh medium. siRNA transfections wereperformed using Lipofectamine™ 2000 (Invitrogen GmbH, Karlsruhe,Germany) as described by the manufacturer. Cells were incubated for 24 hat 37° C. and 5% CO₂ in a humidified incubator (Heraeus GmbH, Hanau,Germany). For the primary screen, all siRNAs were screened at a finalconcentration of 30 nM. Selected sequences were rescreened at a siRNAconcentration of 300 pM. Each siRNA was tested in quadruplicate for eachconcentration.

Cells were lysed by removing growth medium and application of 150 μl ofa 1:1 mixture consisting of medium and substrate from the Dual-GloLuciferase Assay System (Promega, Mannheim, Germany). The luciferaseassay was performed according to the manufacturer's protocol forDual-Glo Luciferase assay and luminescence was measured in aVictor-Light 1420 Luminescence Counter (Perkin Elmer, Rodgau-Jügesheim,Germany). Values obtained with Renilla luciferase were normalized to therespective values obtained with Firefly luciferase in order to correctfor transfection efficacy. Renilla/Firefly luciferase activitiesobtained after transfection with siRNAs directed against a JCV gene werenormalized to Renilla/Firefly luciferase activities obtained aftertransfection of an unrelated control siRNA set to 100%. Tables 1a and bprovides the results where the siRNAs, the sequences of which are givenin Tables 1a and b, were tested at a single dose of 30 nM. Thepercentage inhibition±standard deviation, compared to the unrelatedcontrol siRNA, is indicated in the column ‘Remaining luciferase activity(% of control)’. A number of JCV siRNAs at 30 nM were effective atreducing levels of the targeted mRNA by more than 70% in Cos-7 cells(i.e. remaining luciferase activity was less than 30%).

Selected JCV siRNAs from the single dose screen were furthercharacterized by dose response curves. Transfections of JCV siRNAs forgeneration of dose response curves were performed with the followingsiRNA concentrations according to the above protocol:

from 33 nM in 3-fold dilutions down to 0.005 nM (for fragment L1)

from 24 nM in 4-fold dilutions down to 0.001 nM (for fragment E andfragments LA23 1-700 and LA23 701-1438).

IC50 values were determined by parameterized curve fitting using theprogram XLfit (IDBS, Guildford, Great Britain). Table 2 provides theresults from two independent experiments for 32 selected JCV siRNAs. Themean 1050 from these two independent experiments is shown. Several JCVsiRNAs (AD-12622, AD-12677, AD-12709, AD-12710, AD-12722, AD-12724,AD-12728, AD-12763, AD-12767, AD-12768, AD-12769, AD-12771, AD-12774,AD-12775, AD-12777, AD-12781, AD-12784, AD-12795, AD-12813, AD-12821,AD-12823, AD-12824, AD-12825, AD-12827, AD-12829, AD-12842) wereparticularly potent in this experimental paradigm, and exhibited 1050values between 70 pM and 1 nM.

TABLE 2 IC50s Mean IC50 Duplex name [nM] AD-12599 2.37 AD-12622 0.57AD-12666 3.7 AD-12677 0.49 AD-12709 0.19 AD-12710 0.47 AD-12712 2.33AD-12722 0.12 AD-12724 0.26 AD-12728 0.8 AD-12761 1.2 AD-12763 0.95AD-12767 0.09 AD-12768 0.19 AD-12769 0.35 AD-12771 0.35 AD-12774 0.13AD-12775 0.18 AD-12777 0.17 AD-12778 12.65 AD-12781 0.18 AD-12784 0.44AD-12795 0.65 AD-12813 0.2 AD-12818 1.88 AD-12821 0.07 AD-12823 0.46AD-12824 0.25 AD-12825 0.52 AD-12827 0.15 AD-12829 0.14 AD-12842 0.44

Screen of JCV siRNAs Against Live JC Virus in SVG-A Cells

Cells and Virus

SVG-A cells (human fetal glial cells transformed by SV40 T antigen)obtained from Walter Atwood at Brown University were cultured in Eagle'sMinimum Essential Media (ATCC, Manassas, Va.) supplemented to contain10% fetal bovine serum (FBS) (Omega Scientific, Tarzana, Calif.),Penicillin 100 U/ml, Streptomycin 100 ug/ml (Invitrogen, CarlsbadCalif.) at 37° C. in an atmosphere with 5% CO₂ in a humidified incubator(Heraeus HERAcell, Thermo Electron Corporation, Ashville, N.C.). TheMad-1-SVEΔ strain of JCV obtained from Walter Atwood at Brown Universitywas used in all experiments; viral stocks were prepared using SVG-Acells according to standard published methods (Liu and Atwood,Propagation and assay of the JC Virus, Methods Mol Biol. 2001;165:9-17).

Prophylaxis Assay

SVG-A cells were seeded on glass coverslips in 6-well dishes 24 hoursprior to transfection in the media described above minus antibiotics.Cells were transfected with the indicated concentration of siRNA (10 nM,50 nM, or 100 nM) using Lipofectamine™ 2000 according to themanufacturer's instructions (Invitrogen, Carlsbad, Calif.). Twenty-fourhours post-transfection cells were washed with media containing 2% FBSand then infected with a 1:25 dilution of JCV virus stock (Mad-1-SVEΔstrain) diluted in 2% FBS media. Cells were rocked every 15 minutes byhand several times to get equal virus binding across the entirecoverslip for one hour and then additional 10% FBS media was added andthe infection was allowed to proceed for 72 hours. Seventy two hourspost-infection, cells were fixed in acetone and stained for late viralprotein (VP1) by standard immunofluoresence methods using hybridomasupernatant PAB597 recognizing JCV VP1 (obtained from Walter Atwood atBrown University) with goat anti-mouse Alexa Fluor 488 secondaryantibody (Invitrogen, Carlsbad, Calif.). Infected cells were scored bycounting VP1-immunoreactive cells using a fluorescence microscope(Zeiss, Imager.Z1, Thornwood, N.J.) and data were expressed as thepercentage of infected cells counted for the control coverslipstransfected with Luciferase siRNA. Table 3 shows the results of theprophylaxis assays at different siRNA concentrations (10 nM, 50 nM or100 nM). The VP1 siRNAs were the most potent as a group, followed by theT antigen siRNAs, with the VP2/3 siRNAs being the least potent. The VP1siRNAs most effective in reducing virus were consistently AD-12622,AD-12728, AD-12795, and AD-12842. The most potent T antigen siRNA wasAD-12813.

TABLE 3 Prophylaxis Assay Remaining Virus Duplex Targeted JCV (% ofLuciferase Control) Number Transcript 50 nM 10 nM 100 nM AD-12599 VP179.9 ND ND AD-12709 VP1 46.0 ND ND AD-12710 VP1 25.9 ND ND AD-12784 VP130.9 ND ND AD-12712 VP1 29.7 ND ND AD-12724 VP1 30.5 38.9 25.8 AD-12622VP1 22.9 28.2 9.1 AD-12728 VP1 21.1 22.2 ND AD-12795 VP1 13.6 16.9 8.5AD-12842 VP1 16.0 23.4 12.7 AD-12761 VP1 26.4 52.3 ND AD-12818 VP1 24.050.2 28.0 AD-12666 VP1 54.1 ND ND AD-12763 VP1 39.5 ND ND AD-12722 TAntigen 43.6 82.1 ND AD-12813 T Antigen 21.5 48.8 19.4 AD-12767 TAntigen 37.6 52.2 30.9 AD-12821 T Antigen 33.0 51.2 30.8 AD-12774 TAntigen 74.0 89.2 ND AD-12827 T Antigen 77.0 92.0 ND AD-12775 T Antigen81.6 95.4 ND AD-12777 T Antigen 73.3 93.9 ND AD-12829 T Antigen 78.693.6 ND AD-12781 T Antigen 38.8 62.6 34.4 AD-12768 VP2/3 73.9 92.4 NDAD-12771 VP2/3 51.6 83.6 ND AD-12824 VP2/3 42.1 79.0 43.7 AD-12769 VP2/335.2 78.0 39.7 AD-12823 VP2/3 38.1 78.1 42.0 AD-12677 VP2/3 99.1 102.1ND AD-12825 VP2/3 100.8 99.1 ND ND indicates no data.

Post-Infection Treatment Assay

SVG-A cells were seeded on glass coverslips in 6-well dishes 24 hoursprior to infection in 10% FBS media. Cells were washed with mediacontaining 2% FBS and then infected with a 1:25 dilution of JCV virusstock diluted in 2% FBS media. Cells were rocked by hand approximately8-10 times to get equal virus binding across the entire coverslip every15 minutes for one hour and then additional 10% FBS media was added.Twenty-four and forty-eight hours postinfection, cells were washed with10% FBS media containing no antibiotics and then transfected with 50 nMof the indicated siRNA using Lipofectamine™ 2000 according to themanufacturer's instructions (Invitrogen, Carlsbad, Calif.). Seventy-twohours postinfection, cells were fixed in acetone and stained for lateviral protein (VP1) by standard immunofluoresence methods usinghybridoma supernatant PAB597 recognizing JCV VP1 (obtained from WalterAtwood at Brown University) with goat anti-mouse Alexa Fluor 488secondary antibody (Molecular Probes, Eugene, Oreg.). Infected cellswere scored by counting VP1-immunoreactive cells using a fluorescencemicroscope (Zeiss, Imager.Z1, Thornwood, N.J.) and data were expressedas the percentage of infected cells counted for control coverslipstransfected with Luciferase siRNA. Table 4 shows the results of thepost-infection treatment experiments. All of the siRNAs tested in thetreatment assay showed significant antiviral activity against JCV, suchthat the remaining virus was significantly less than that in theluciferase siRNA control.

TABLE 4 Treatment Assay Targeted JCV Remaining Virus Duplex NumberTranscript (% of Luciferase Control) AD-12724 VP1 38.9 AD-12622 VP1 28.2AD-12795 VP1 16.9 AD-12842 VP1 23.4 AD-12818 VP1 ND AD-12813 T Antigen48 AD-12767 T Antigen 56.9 AD-12821 T Antigen 75.8 AD-12781 T Antigen75.8 AD-12824 VP2/3 60.4 AD-12769 VP2/3 70.7 AD-12823 VP2/3 72.4 NDindicates no data.

Prophylaxis Administration of JCV siRNAs Inhibits the Production ofActive Progeny JC Virus

SVG-A cells were seeded in 6-well dishes 24 hours prior to transfectionin the media described above minus antibiotics. Cells were transfectedwith 10 nM of the indicated siRNA using Lipofectamine™ 2000 according tothe manufacturer's instructions (Invitrogen, Carlsbad, Calif.).Twenty-four hours post-transfection cells were washed with mediacontaining 2% FBS and then infected with a 1:25 dilution of JCV virusstock (Mad-1-SVEΔ strain) diluted in 2% FBS media. Cells were rockedevery 15 minutes by hand several times to get equal virus binding acrossthe entire coverslip for one hour and then additional 10% FBS media wasadded and the infection was allowed to proceed for 6 days. Six dayspost-infection, progeny virus was collected either by removal of overlaymedia from infected cells or by scraping cells and performing viruspreparations. The virus preparations consisted of scraping cells intothe supernatant media, vortexing, freeze-thawing the re-suspended cells2 times with vortexing in between, then spinning down the cell debrisand taking the supernatant. Fresh SVG-A cells seeded on glass coverslipswere infected secondarily with virus collected by either method usingthe same procedure done with the initial infection to determine theamount of infectious virus produced by cells transfected with thevarious siRNAs. At 72 hours post-infection of coverslips, cells werefixed in acetone and stained for late viral protein (VP1) by standardimmunofluoresence methods using hybridoma supernatant PAB597 recognizingJCV VP1 (obtained from Walter Atwood at Brown University) with goatanti-mouse Alexa Fluor 488 secondary antibody (Invitrogen, Carlsbad,Calif.). Infected cells were scored by counting VP1-immunoreactive cellsusing a fluorescence microscope (Zeiss, Imager.Z1, Thornwood, N.J.) anddata were expressed as the percentage of infected cells counted for thecontrol coverslips transfected with Luciferase siRNA. Table 5 shows theresults for selected siRNAs, demonstrating the ability of prophylaxissiRNA treatment to inhibit active progeny virus production by eithermethod of virus collection. Transfection with siRNAs targeting VP1(AD-12622 and AD-12842) had the greatest effect on inhibiting theproduction of active progeny virus regardless of whether virus wascollected from media or from infected cell preparations. The T antigensiRNA AD-12813 had the next strongest inhibitory effect, whereas theVP2/3 siRNAs AD-12824 and AD-12769 still showed some albeit a lesserability to inhibit active progeny JCV production.

TABLE 5 Prophylaxis administration of JCV siRNAs inhibits the productionof active progeny JC virus capable of secondary infection RemainingVirus (% of Luciferase Control) Virus Duplex Name Targeted TranscriptMedia Preparation AD-12622 VP1 30.8 24.9 AD-12842 VP1 33.3 26.9 AD-12813T Antigen 57.8 38.7 AD-12824 VP2/3 83.6 57.6 AD-12769 VP2/3 79.1 52.2

Stability in Cerebrospinal Fluid (CSF) of Selected siRNAs Targeting JCV

Eleven selected JCV siRNAs were tested for stability at 5 uM over 48 hat 37° C. in human CSF, as well as in PBS for comparison. 30 μl of humancerebrospinal fluid (CSF) was mixed with 3 μl of 50 μM duplex (siRNA)solution (150 pmole/well) in a 96-well plate, sealed to avoidevaporation and incubated for the indicated time at 37° C. Incubation ofthe siRNA in 30 ul PBS for 48 h served as a control for non-specificdegradation. Reactions were stopped by the addition of 4 ul proteinase K(20 mg/ml) and 25 ul of proteinase K buffer, and an incubation for 20′at 42° C. Samples were then spin filtered through a 0.2 μm 96 wellfilter plate at 3000 rpm for 20′. Incubation wells were washed with 50ul Millipore water twice and the combined washing solutions were spinfiltered also.

Samples were analyzed by ion exchange HPLC under denaturing conditions.Samples were transferred to single autosampler vials. IEX-HPLC analysiswas performed under the following conditions: Dionex DNAPac PA200 (4×250mm analytical column), temperature of 45° C. (denaturing conditions bypH=11), flow rate of 1 ml/min, injection volume of 50 ul, and detectionwavelength of 260 nm with 1 nm bandwidth (reference wavelength 600 nm).In addition, the gradient conditions were as follows with HPLC Eluent A:20 mM Na₃PO₄ in 10% ACN; pH=11 and HPLC Eluent B: 1 M NaBr in HPLCEluent A:

Time % A % B 0.00 min 75 25 1.00 min 75 25 19.0 min 38 62 19.5 min 0 10021.5 min 0 100 22.0 min 75 25 24.0 min 75 25

Under the above denaturing IEX-HPLC conditions, the duplexes eluted astwo separated single strands. All chromatograms were integratedautomatically by the Dionex Chromeleon 6.60 HPLC software, and wereadjusted manually as necessary. The area under the peak for each strandwas calculated and the %-values for each intact full length product(FLP) for each time points were calculated by the following equation:

%-FLP_((s/as; t=x))=(PeakArea_((s/as); t=x)/PeakArea_((s/as); t=0 min))*100%

All values were normalized to FLP at t=0 min. Table 6 provides theresults after 48 hours of incubation in human CSF at 37° C. At least 75%of both antisense and sense strands of ten JCV siRNAs (AD-12622,AD-12724, AD-12767, AD-12769, AD-12795, AD-12813, AD-12818, AD-12823,AD-12824, AD-12842) were recovered, demonstrating that these siRNAs arehighly stable in human CSF at 37° C. For AD-12821, 59% of the antisenseand 97% of the sense strand was recovered after 48 h of incubation inhuman CSF at 37° C., showing that this siRNA has a half-life of greaterthan 48 h in human CSF at 37° C.

TABLE 6 Stability in human CSF Duplex % full length material after 48hours name antisense sense AD-12622 93 105 AD-12724 90 106 AD-12767 85104 AD-12769 100 104 AD-12795 86 109 AD-12813 94 98 AD-12818 75 99AD-12821 59 97 AD-12823 98 98 AD-12824 84 98 AD-12842 87 102

dsRNA Expression Vectors

In another aspect of the invention, JC virus specific dsRNA moleculesthat modulate JC virus genome expression activity are expressed fromtranscription units inserted into DNA or RNA vectors (see, e.g.,Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al.,International PCT Publication No. WO 00/22113, Conrad, International PCTPublication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Thesetransgenes can be introduced as a linear construct, a circular plasmid,or a viral vector, which can be incorporated and inherited as atransgene integrated into the host genome. The transgene can also beconstructed to permit it to be inherited as an extrachromosomal plasmid(Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on twoseparate expression vectors and co-transfected into a target cell.Alternatively each individual strand of the dsRNA can be transcribed bypromoters both of which are located on the same expression plasmid. In apreferred embodiment, a dsRNA is expressed as an inverted repeat joinedby a linker polynucleotide sequence such that the dsRNA has a stem andloop structure.

The recombinant dsRNA expression vectors are generally DNA plasmids orviral vectors. dsRNA expressing viral vectors can be constructed basedon, but not limited to, adeno-associated virus (for a review, seeMuzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129));adenovirus (see, for example, Berkner, et al., BioTechniques (1998)6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld etal. (1992), Cell 68:143-155)); or alphavirus as well as others known inthe art. Retroviruses have been used to introduce a variety of genesinto many different cell types, including epithelial cells, in vitroand/or in vivo (see, e.g., Eglitis, et al., Science (1985)230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998)85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem. et al., 1992, Proc. Natl. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Recombinant retroviralvectors capable of transducing and expressing genes inserted into thegenome of a cell can be produced by transfecting the recombinantretroviral genome into suitable packaging cell lines such as PA317 andPsi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al.,1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviralvectors can be used to infect a wide variety of cells and tissues insusceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al.,1992, J. Infectious Disease, 166:769), and also have the advantage ofnot requiring mitotically active cells for infection.

The promoter driving dsRNA expression in either a DNA plasmid or viralvector of the invention may be a eukaryotic RNA polymerase I (e.g.ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter oractin promoter or U1 snRNA promoter) or generally RNA polymerase IIIpromoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter,for example the T7 promoter, provided the expression plasmid alsoencodes T7 RNA polymerase required for transcription from a T7 promoter.The promoter can also direct transgene expression to the pancreas (see,e.g. the insulin regulatory sequence for pancreas (Bucchini et al.,1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals include regulation by ecdysone, by estrogen, progesterone,tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules aredelivered as described below, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of dsRNA molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the dsRNAs bind to target RNAand modulate its function or expression. Delivery of dsRNA expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from thepatient followed by reintroduction into the patient, or by any othermeans that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into targetcells as a complex with cationic lipid carriers (e.g. Oligofectamine) ornon-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipidtransfections for dsRNA-mediated knockdowns targeting different regionsof a single JC virus genome or multiple JC virus genomes over a periodof a week or more are also contemplated by the invention. Successfulintroduction of the vectors of the invention into host cells can bemonitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection ofex vivo cells can be ensured using markers that provide the transfectedcell with resistance to specific environmental factors (e.g.,antibiotics and drugs), such as hygromycin B resistance.

The JC virus specific dsRNA molecules can also be inserted into vectorsand used as gene therapy vectors for human patients. Gene therapyvectors can be delivered to a subject by, for example, intravenousinjection, local administration (see U.S. Pat. No. 5,328,470) or bystereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad.Sci. USA 91:3054-3057). The pharmaceutical preparation of the genetherapy vector can include the gene therapy vector in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle is imbedded. Alternatively, where the complete genedelivery vector can be produced intact from recombinant cells, e.g.,retroviral vectors, the pharmaceutical preparation can include one ormore cells which produce the gene delivery system.

Tables 1a-1 and 1a-2

TABLE 1a JCV Gene Walk; siRNAs targeting >95% of all stransins (> =out of 388); Human specific pan-JCV: 208 siRNAs;all siRNAs double overhang design; dTdT, nomodifications. 1 a-1: sequences; 1 a-2: assay results Table 1a-1 SEQ SEQduplex position in ID sense strand ID antisense strand name consensusNO: sequence (5′-3′) NO: sequence (5′-3′) AD-14742 1533-1551 515CUUAUAAGAGGAGGAGUAGTT 516 CUACUCCUCCUCUUAUAAGTT AD-14743 1703-1721 517CAUGCUUCCUUGUUACAGUTT 518 ACUGUAACAAGGAAGCAUGTT AD-14744 1439-1457 519UACGGGACUGUAACACCUGTT 520 CAGGUGUUACAGUCCCGUATT AD-14745 1705-1723 521UGCUUCCUUGUUACAGUGUTT 522 ACACUGUAACAAGGAAGCATT AD-14746 2064-2082 523CCUGUUGAAUGUUGGGUUCTT 524 GAACCCAACAUUCAACAGGTT AD-14747 2067-2085 525GUUGAAUGUUGGGUUCCUGTT 526 CAGGAACCCAACAUUCAACTT AD-14748 2071-2089 527AAUGUUGGGUUCCUGAUCCTT 528 GGAUCAGGAACCCAACAUUTT AD-14749 2121-2139 529ACACUAACAGGAGGAGAAATT 530 UUUCUCCUCCUGUUAGUGUTT AD-14750 1535-1553 531UAUAAGAGGAGGAGUAGAATT 532 UUCUACUCCUCCUCUUAUATT AD-14751 1536-1554 533AUAAGAGGAGGAGUAGAAGTT 534 CUUCUACUCCUCCUCUUAUTT AD-14752 1445-1463 535ACUGUAACACCUGCUCUUGTT 536 CAAGAGCAGGUGUUACAGUTT AD-14753 1700-1718 537GGACAUGCUUCCUUGUUACTT 538 GUAACAAGGAAGCAUGUCCTT AD-14754 1702-1720 539ACAUGCUUCCUUGUUACAGTT 540 CUGUAACAAGGAAGCAUGUTT AD-14755 1704-1722 541AUGCUUCCUUGUUACAGUGTT 542 CACUGUAACAAGGAAGCAUTT AD-14756 2065-2083 543CUGUUGAAUGUUGGGUUCCTT 544 GGAACCCAACAUUCAACAGTT AD-14757 2070-2088 545GAAUGUUGGGUUCCUGAUCTT 546 GAUCAGGAACCCAACAUUCTT AD-14758 1441-1459 547CGGGACUGUAACACCUGCUTT 548 AGCAGGUGUUACAGUCCCGTT AD-14759 1443-1461 549GGACUGUAACACCUGCUCUTT 550 AGAGCAGGUGUUACAGUCCTT AD-14760 1444-1462 551GACUGUAACACCUGCUCUUTT 552 AAGAGCAGGUGUUACAGUCTT AD-14761 1609-1627 553CUCCAGAAAUGGGUGACCCTT 554 GGGUCACCCAUUUCUGGAGTT AD-14762 1537-1555 555UAAGAGGAGGAGUAGAAGUTT 556 ACUUCUACUCCUCCUCUUATT AD-14763 629-647 557GAGGCUGCUGCUACUAUAGTT 558 CUAUAGUAGCAGCAGCCUCTT AD-14764 656-674 559AUUGCAUCCCUUGCUACUGTT 560 CAGUAGCAAGGGAUGCAAUTT AD-14765 658-676 561UGCAUCCCUUGCUACUGUATT 562 UACAGUAGCAAGGGAUGCATT AD-14766 517-535 563UUGUGUUUUCAGGUUCAUGTT 564 CAUGAACCUGAAAACACAATT AD-14767 559-577 565GGACCUAGUUGCUACUGUUTT 566 AACAGUAGCAACUAGGUCCTT AD-14768 591-609 567CUGCCACAGGAUUUUCAGUTT 568 ACUGAAAAUCCUGUGGCAGTT AD-14769 638-656 569GCUACUAUAGAAGUUGAAATT 570 UUUCAACUUCUAUAGUAGCTT AD-14770 655-673 571AAUUGCAUCCCUUGCUACUTT 572 AGUAGCAAGGGAUGCAAUUTT AD-14771 561-579 573ACCUAGUUGCUACUGUUUCTT 574 GAAACAGUAGCAACUAGGUTT AD-14772 639-657 575CUACUAUAGAAGUUGAAAUTT 576 AUUUCAACUUCUAUAGUAGTT AD-14773 715-733 577AGGCCUUACUCCUGAAACATT 578 UGUUUCAGGAGUAAGGCCUTT AD-14774 716-734 579GGCCUUACUCCUGAAACAUTT 580 AUGUUUCAGGAGUAAGGCCTT AD-14775 326-344 581GUAAAACCUGGAGUGGAACTT 582 GUUCCACUCCAGGUUUUACTT AD-14776 518-536 583UGUGUUUUCAGGUUCAUGGTT 584 CCAUGAACCUGAAAACACATT AD-14777 520-538 585UGUUUUCAGGUUCAUGGGUTT 586 ACCCAUGAACCUGAAAACATT AD-14778 661-679 587AUCCCUUGCUACUGUAGAGTT 588 CUCUACAGUAGCAAGGGAUTT AD-14779 560-578 589GACCUAGUUGCUACUGUUUTT 590 AAACAGUAGCAACUAGGUCTT AD-14780 681-699 591GGAUUACAAGUACCUCUGATT 592 UCAGAGGUACUUGUAAUCCTT AD-14781 714-732 593UAGGCCUUACUCCUGAAACTT 594 GUUUCAGGAGUAAGGCCUATT AD-14782 377-395 595UGUUAGAAUUUUUGCUGGATT 596 UCCAGCAAAAAUUCUAACATT AD-14783 589-607 597UGCUGCCACAGGAUUUUCATT 598 UGAAAAUCCUGUGGCAGCATT AD-14784 594-612 599CCACAGGAUUUUCAGUAGCTT 600 GCUACUGAAAAUCCUGUGGTT AD-14785 648-666 601AAGUUGAAAUUGCAUCCCUTT 602 AGGGAUGCAAUUUCAACUUTT AD-14786 649-667 603AGUUGAAAUUGCAUCCCUUTT 604 AAGGGAUGCAAUUUCAACUTT AD-14787 587-605 605GCUGCUGCCACAGGAUUUUTT 606 AAAAUCCUGUGGCAGCAGCTT AD-14788 325-343 607AGUAAAACCUGGAGUGGAATT 608 UUCCACUCCAGGUUUUACUTT AD-14789 515-533 609UUUUGUGUUUUCAGGUUCATT 610 UGAACCUGAAAACACAAAATT AD-14790 516-534 611UUUGUGUUUUCAGGUUCAUTT 612 AUGAACCUGAAAACACAAATT AD-14791 519-537 613GUGUUUUCAGGUUCAUGGGTT 614 CCCAUGAACCUGAAAACACTT AD-14792 521-539 615GUUUUCAGGUUCAUGGGUGTT 616 CACCCAUGAACCUGAAAACTT AD-14793 522-540 617UUUUCAGGUUCAUGGGUGCTT 618 GCACCCAUGAACCUGAAAATT AD-14794 523-541 619UUUCAGGUUCAUGGGUGCCTT 620 GGCACCCAUGAACCUGAAATT AD-14795 616-634 621AAUUGCUGCUGGAGAGGCUTT 622 AGCCUCUCCAGCAGCAAUUTT AD-14796 657-675 623UUGCAUCCCUUGCUACUGUTT 624 ACAGUAGCAAGGGAUGCAATT AD-14797 761-779 625GCUGUAGCUGGGUUUGCUGTT 626 CAGCAAACCCAGCUACAGCTT AD-14798 645-663 627UAGAAGUUGAAAUUGCAUCTT 628 GAUGCAAUUUCAACUUCUATT AD-14799 647-665 629GAAGUUGAAAUUGCAUCCCTT 630 GGGAUGCAAUUUCAACUUCTT AD-14800 660-678 631CAUCCCUUGCUACUGUAGATT 632 UCUACAGUAGCAAGGGAUGTT AD-14801 324-342 633UAGUAAAACCUGGAGUGGATT 634 UCCACUCCAGGUUUUACUATT AD-14802 372-390 635UUUUUUGUUAGAAUUUUUGTT 636 CAAAAAUUCUAACAAAAAATT AD-14803 640-658 637UACUAUAGAAGUUGAAAUUTT 638 AAUUUCAACUUCUAUAGUATT AD-14804 562-580 639CCUAGUUGCUACUGUUUCUTT 640 AGAAACAGUAGCAACUAGGTT AD-14805 563-581 641CUAGUUGCUACUGUUUCUGTT 642 CAGAAACAGUAGCAACUAGTT AD-14806 566-584 643GUUGCUACUGUUUCUGAGGTT 644 CCUCAGAAACAGUAGCAACTT AD-14807 625-643 645UGGAGAGGCUGCUGCUACUTT 646 AGUAGCAGCAGCCUCUCCATT AD-14808 627-645 647GAGAGGCUGCUGCUACUAUTT 648 AUAGUAGCAGCAGCCUCUCTT AD-14809 628-646 649AGAGGCUGCUGCUACUAUATT 650 UAUAGUAGCAGCAGCCUCUTT AD-14810 632-650 651GCUGCUGCUACUAUAGAAGTT 652 CUUCUAUAGUAGCAGCAGCTT AD-14811 513-531 653UUUUUUGUGUUUUCAGGUUTT 654 AACCUGAAAACACAAAAAATT AD-14812 641-659 655ACUAUAGAAGUUGAAAUUGTT 656 CAAUUUCAACUUCUAUAGUTT AD-14813 323-341 657UUAGUAAAACCUGGAGUGGTT 658 CCACUCCAGGUUUUACUAATT AD-14814 717-735 659GCCUUACUCCUGAAACAUATT 660 UAUGUUUCAGGAGUAAGGCTT AD-14815 646-664 661AGAAGUUGAAAUUGCAUCCTT 662 GGAUGCAAUUUCAACUUCUTT AD-14816 592-610 663UGCCACAGGAUUUUCAGUATT 664 UACUGAAAAUCCUGUGGCATT AD-14817 590-608 665GCUGCCACAGGAUUUUCAGTT 666 CUGAAAAUCCUGUGGCAGCTT AD-14818 526-544 667CAGGUUCAUGGGUGCCGCATT 668 UGCGGCACCCAUGAACCUGTT AD-14819 615-633 669AAAUUGCUGCUGGAGAGGCTT 670 GCCUCUCCAGCAGCAAUUUTT AD-14820 617-635 671AUUGCUGCUGGAGAGGCUGTT 672 CAGCCUCUCCAGCAGCAAUTT AD-14821 652-670 673UGAAAUUGCAUCCCUUGCUTT 674 AGCAAGGGAUGCAAUUUCATT AD-14822 374-392 675UUUUGUUAGAAUUUUUGCUTT 676 AGCAAAAAUUCUAACAAAATT AD-14823 375-393 677UUUGUUAGAAUUUUUGCUGTT 678 CAGCAAAAAUUCUAACAAATT AD-14824 631-649 679GGCUGCUGCUACUAUAGAATT 680 UUCUAUAGUAGCAGCAGCCTT AD-14825 376-394 681UUGUUAGAAUUUUUGCUGGTT 682 CCAGCAAAAAUUCUAACAATT AD-14826 512-530 683UUUUUUUGUGUUUUCAGGUTT 684 ACCUGAAAACACAAAAAAATT AD-14827 1127-1145 685GAAACUACUUGGGCAAUAGTT 686 CUAUUGCCCAAGUAGUUUCTT AD-14828 1410-1428 687AAUGGAUGUUGCCUUUACUTT 688 AGUAAAGGCAACAUCCAUUTT AD-14829 1406-1424 689CCUCAAUGGAUGUUGCCUUTT 690 AAGGCAACAUCCAUUGAGGTT AD-14830 1418-1436 691UUGCCUUUACUUUUAGGGUTT 692 ACCCUAAAAGUAAAGGCAATT AD-14831 1126-1144 693AGAAACUACUUGGGCAAUATT 694 UAUUGCCCAAGUAGUUUCUTT AD-14832 1125-1143 695AAGAAACUACUUGGGCAAUTT 696 AUUGCCCAAGUAGUUUCUUTT AD-14833 1419-1437 697UGCCUUUACUUUUAGGGUUTT 698 AACCCUAAAAGUAAAGGCATT AD-14834 1420-1438 699GCCUUUACUUUUAGGGUUGTT 700 CAACCCUAAAAGUAAAGGCTT AD-14835 1422-1440 701CUUUACUUUUAGGGUUGUATT 702 UACAACCCUAAAAGUAAAGTT AD-14836 1423-1441 703UUUACUUUUAGGGUUGUACTT 704 GUACAACCCUAAAAGUAAATT AD-14837 1425-1443 705UACUUUUAGGGUUGUACGGTT 706 CCGUACAACCCUAAAAGUATT AD-14838 1123-1141 707GGAAGAAACUACUUGGGCATT 708 UGCCCAAGUAGUUUCUUCCTT AD-14839 1409-1427 709CAAUGGAUGUUGCCUUUACTT 710 GUAAAGGCAACAUCCAUUGTT AD-14840 1413-1431 711GGAUGUUGCCUUUACUUUUTT 712 AAAAGUAAAGGCAACAUCCTT AD-14841 1416-1434 713UGUUGCCUUUACUUUUAGGTT 714 CCUAAAAGUAAAGGCAACATT AD-14842 1414-1432 715GAUGUUGCCUUUACUUUUATT 716 UAAAAGUAAAGGCAACAUCTT AD-14843 911-929 717CCAGAAGACUACUAUGAUATT 718 UAUCAUAGUAGUCUUCUGGTT AD-14844 910-928 719UCCAGAAGACUACUAUGAUTT 720 AUCAUAGUAGUCUUCUGGATT AD-14845 1120-1138 721UUUGGAAGAAACUACUUGGTT 722 CCAAGUAGUUUCUUCCAAATT AD-14846 1404-1422 723CUCCUCAAUGGAUGUUGCCTT 724 GGCAACAUCCAUUGAGGAGTT AD-14847 1337-1355 725CCAAAUGUGCAAUCUGGUGTT 726 CACCAGAUUGCACAUUUGGTT AD-14848 1338-1356 727CAAAUGUGCAAUCUGGUGATT 728 UCACCAGAUUGCACAUUUGTT AD-14849 1397-1415 729AGAUCUGCUCCUCAAUGGATT 730 UCCAUUGAGGAGCAGAUCUTT AD-14850 1407-1425 731CUCAAUGGAUGUUGCCUUUTT 732 AAAGGCAACAUCCAUUGAGTT AD-14851 4157-4175 733GCUCAAAUUUUAUAUAAGATT 734 UCUUAUAUAAAAUUUGAGCTT AD-14852 4795-4813 735AGCCUGAUUUUGGUACAUGTT 736 CAUGUACCAAAAUCAGGCUTT AD-14853 4156-4174 737CUCAAAUUUUAUAUAAGAATT 738 UUCUUAUAUAAAAUUUGAGTT AD-14854 5002-5020 739ACAAAGUGCUGAAUAGGGATT 740 UCCCUAUUCAGCACUUUGUTT AD-14855 4792-4810 741CUGAUUUUGGUACAUGGAATT 742 UUCCAUGUACCAAAAUCAGTT AD-14856 4790-4808 743GAUUUUGGUACAUGGAAUATT 744 UAUUCCAUGUACCAAAAUCTT AD-14857 4801-4819 745CUCAUCAGCCUGAUUUUGGTT 746 CCAAAAUCAGGCUGAUGAGTT AD-14858 4622-4640 747AGCCCACUUGUGUGGAUAGTT 748 CUAUCCACACAAGUGGGCUTT AD-14859 4997-5015 749GUGCUGAAUAGGGAGGAAUTT 750 AUUCCUCCCUAUUCAGCACTT AD-14860 5094-5112 751AGUAAGGGCGUGGAGGCUUTT 752 AAGCCUCCACGCCCUUACUTT AD-14861 4564-4582 753GUGACUUAACCCAAGAAGCTT 754 GCUUCUUGGGUUAAGUCACTT AD-14862 5095-5113 755UAGUAAGGGCGUGGAGGCUTT 756 AGCCUCCACGCCCUUACUATT AD-14863 4800-4818 757UCAUCAGCCUGAUUUUGGUTT 758 ACCAAAAUCAGGCUGAUGATT AD-14864 4265-4283 759GUAGAAGACCCUAAAGACUTT 760 AGUCUUUAGGGUCUUCUACTT AD-14865 4267-4285 761AGGUAGAAGACCCUAAAGATT 762 UCUUUAGGGUCUUCUACCUTT AD-14866 4270-4288 763AAAAGGUAGAAGACCCUAATT 764 UUAGGGUCUUCUACCUUUUTT AD-14867 4269-4287 765AAAGGUAGAAGACCCUAAATT 766 UUUAGGGUCUUCUACCUUUTT AD-14868 2874-2892 767GAUUGUGCAGUGGAAAGAATT 768 UUCUUUCCACUGCACAAUCTT AD-14869 2875-2893 769GGAUUGUGCAGUGGAAAGATT 770 UCUUUCCACUGCACAAUCCTT AD-14870 3950-3968 771UGUAGACAGCCAUAUGCAGTT 772 CUGCAUAUGGCUGUCUACATT AD-14871 3896-3914 773CAUGACUUUAACCCAGAAGTT 774 CUUCUGGGUUAAAGUCAUGTT AD-14872 4990-5008 775AUAGGGAGGAAUCCAUGGATT 776 UCCAUGGAUUCCUCCCUAUTT AD-14873 4994-5012 777CUGAAUAGGGAGGAAUCCATT 778 UGGAUUCCUCCCUAUUCAGTT AD-14874 5000-5018 779AAAGUGCUGAAUAGGGAGGTT 780 CCUCCCUAUUCAGCACUUUTT AD-14875 4563-4581 781UGACUUAACCCAAGAAGCUTT 782 AGCUUCUUGGGUUAAGUCATT AD-14876 3895-3913 783AUGACUUUAACCCAGAAGATT 784 UCUUCUGGGUUAAAGUCAUTT AD-14877 4262-4280 785GAAGACCCUAAAGACUUUCTT 786 GAAAGUCUUUAGGGUCUUCTT AD-14878 4162-4180 787AAAAAGCUCAAAUUUUAUATT 788 UAUAAAAUUUGAGCUUUUUTT AD-14879 4798-4816 789AUCAGCCUGAUUUUGGUACTT 790 GUACCAAAAUCAGGCUGAUTT AD-14880 4799-4817 791CAUCAGCCUGAUUUUGGUATT 792 UACCAAAAUCAGGCUGAUGTT AD-14881 5006-5024 793AUGGACAAAGUGCUGAAUATT 794 UAUUCAGCACUUUGUCCAUTT AD-14882 4264-4282 795UAGAAGACCCUAAAGACUUTT 796 AAGUCUUUAGGGUCUUCUATT AD-14883 4268-4286 797AAGGUAGAAGACCCUAAAGTT 798 CUUUAGGGUCUUCUACCUUTT AD-14884 4623-4641 799CAGCCCACUUGUGUGGAUATT 800 UAUCCACACAAGUGGGCUGTT AD-14885 4788-4806 801UUUUGGUACAUGGAAUAGUTT 802 ACUAUUCCAUGUACCAAAATT AD-14886 4993-5011 803UGAAUAGGGAGGAAUCCAUTT 804 AUGGAUUCCUCCCUAUUCATT AD-14887 4995-5013 805GCUGAAUAGGGAGGAAUCCTT 806 GGAUUCCUCCCUAUUCAGCTT AD-14888 4996-5014 807UGCUGAAUAGGGAGGAAUCTT 808 GAUUCCUCCCUAUUCAGCATT AD-14889 3952-3970 809UGUGUAGACAGCCAUAUGCTT 810 GCAUAUGGCUGUCUACACATT AD-14890 4595-4613 811UGCUUUGAUUGCUUCAGACTT 812 GUCUGAAGCAAUCAAAGCATT AD-14891 4596-4614 813UUGCUUUGAUUGCUUCAGATT 814 UCUGAAGCAAUCAAAGCAATT AD-14892 4597-4615 815AUUGCUUUGAUUGCUUCAGTT 816 CUGAAGCAAUCAAAGCAAUTT AD-14893 4599-4617 817CUAUUGCUUUGAUUGCUUCTT 818 GAAGCAAUCAAAGCAAUAGTT AD-14894 4726-4744 819AUUGCAAGGAAUGGCCUAATT 820 UUAGGCCAUUCCUUGCAAUTT AD-14895 4753-4771 821AUUUUCCUCCUAAUUCUGATT 822 UCAGAAUUAGGAGGAAAAUTT AD-14896 4802-4820 823GCUCAUCAGCCUGAUUUUGTT 824 CAAAAUCAGGCUGAUGAGCTT AD-14897 4803-4821 825UGCUCAUCAGCCUGAUUUUTT 826 AAAAUCAGGCUGAUGAGCATT AD-14898 4806-4824 827AGUUGCUCAUCAGCCUGAUTT 828 AUCAGGCUGAUGAGCAACUTT AD-14899 5091-5109 829AAGGGCGUGGAGGCUUUUUTT 830 AAAAAGCCUCCACGCCCUUTT AD-14900 5093-5111 831GUAAGGGCGUGGAGGCUUUTT 832 AAAGCCUCCACGCCCUUACTT AD-14901 4259-4277 833GACCCUAAAGACUUUCCUGTT 834 CAGGAAAGUCUUUAGGGUCTT AD-14902 3901-3919 835AGGAGCAUGACUUUAACCCTT 836 GGGUUAAAGUCAUGCUCCUTT AD-14903 4757-4775 837UGUGAUUUUCCUCCUAAUUTT 838 AAUUAGGAGGAAAAUCACATT AD-14904 4758-4776 839UUGUGAUUUUCCUCCUAAUTT 840 AUUAGGAGGAAAAUCACAATT AD-14905 4562-4580 841GACUUAACCCAAGAAGCUCTT 842 GAGCUUCUUGGGUUAAGUCTT AD-14906 4585-4603 843GCUUCAGACAAUGGUUUGGTT 844 CCAAACCAUUGUCUGAAGCTT AD-14907 4587-4605 845UUGCUUCAGACAAUGGUUUTT 846 AAACCAUUGUCUGAAGCAATT AD-14908 4588-4606 847AUUGCUUCAGACAAUGGUUTT 848 AACCAUUGUCUGAAGCAAUTT AD-14909 4591-4609 849UUGAUUGCUUCAGACAAUGTT 850 CAUUGUCUGAAGCAAUCAATT AD-14910 5003-5021 851GACAAAGUGCUGAAUAGGGTT 852 CCCUAUUCAGCACUUUGUCTT AD-14911 4165-4183 853AAGAAAAAGCUCAAAUUUUTT 854 AAAAUUUGAGCUUUUUCUUTT AD-14912 4166-4184 855AAAGAAAAAGCUCAAAUUUTT 856 AAAUUUGAGCUUUUUCUUUTT AD-14913 4263-4281 857AGAAGACCCUAAAGACUUUTT 858 AAAGUCUUUAGGGUCUUCUTT AD-14914 4274-4292 859AAAAAAAAGGUAGAAGACCTT 860 GGUCUUCUACCUUUUUUUUTT AD-14915 4266-4284 861GGUAGAAGACCCUAAAGACTT 862 GUCUUUAGGGUCUUCUACCTT AD-14916 4272-4290 863AAAAAAGGUAGAAGACCCUTT 864 AGGGUCUUCUACCUUUUUUTT AD-14917 4271-4289 865AAAAAGGUAGAAGACCCUATT 866 UAGGGUCUUCUACCUUUUUTT AD-14918 4559-4577 867UUAACCCAAGAAGCUCUUCTT 868 GAAGAGCUUCUUGGGUUAATT AD-14919 4789-4807 869AUUUUGGUACAUGGAAUAGTT 870 CUAUUCCAUGUACCAAAAUTT AD-14920 4998-5016 871AGUGCUGAAUAGGGAGGAATT 872 UUCCUCCCUAUUCAGCACUTT AD-14921 5070-5088 873GAGGCCAGGGAAAUUCCCUTT 874 AGGGAAUUUCCCUGGCCUCTT AD-14922 4158-4176 875AGCUCAAAUUUUAUAUAAGTT 876 CUUAUAUAAAAUUUGAGCUTT AD-14923 5065-5083 877CAGGGAAAUUCCCUUGUUUTT 878 AAACAAGGGAAUUUCCCUGTT AD-14924 2872-2890 879UUGUGCAGUGGAAAGAAAGTT 880 CUUUCUUUCCACUGCACAATT AD-14925 4782-4800 881UACAUGGAAUAGUUCAGAGTT 882 CUCUGAACUAUUCCAUGUATT AD-14926 4783-4801 883GUACAUGGAAUAGUUCAGATT 884 UCUGAACUAUUCCAUGUACTT AD-14927 5064-5082 885AGGGAAAUUCCCUUGUUUUTT 886 AAAACAAGGGAAUUUCCCUTT AD-14928 5071-5089 887GGAGGCCAGGGAAAUUCCCTT 888 GGGAAUUUCCCUGGCCUCCTT AD-14929 3951-3969 889GUGUAGACAGCCAUAUGCATT 890 UGCAUAUGGCUGUCUACACTT AD-14930 3949-3967 891GUAGACAGCCAUAUGCAGUTT 892 ACUGCAUAUGGCUGUCUACTT AD-14931 4355-4373 893GAAGACCUGUUUUGCCAUGTT 894 CAUGGCAAAACAGGUCUUCTT AD-14932 4363-4381 895AGUGGGAUGAAGACCUGUUTT 896 AACAGGUCUUCAUCCCACUTT AD-14933 4356-4374 897UGAAGACCUGUUUUGCCAUTT 898 AUGGCAAAACAGGUCUUCATT AD-14934 4361-4379 899UGGGAUGAAGACCUGUUUUTT 900 AAAACAGGUCUUCAUCCCATT AD-14935 4560-4578 901CUUAACCCAAGAAGCUCUUTT 902 AAGAGCUUCUUGGGUUAAGTT AD-14936 2873-2891 903AUUGUGCAGUGGAAAGAAATT 904 UUUCUUUCCACUGCACAAUTT AD-14937 4730-4748 905CUUUAUUGCAAGGAAUGGCTT 906 GCCAUUCCUUGCAAUAAAGTT AD-14938 3899-3917 907GAGCAUGACUUUAACCCAGTT 908 CUGGGUUAAAGUCAUGCUCTT AD-14939 4756-4774 909GUGAUUUUCCUCCUAAUUCTT 910 GAAUUAGGAGGAAAAUCACTT AD-14940 4590-4608 911UGAUUGCUUCAGACAAUGGTT 912 CCAUUGUCUGAAGCAAUCATT AD-14941 4159-4177 913AAGCUCAAAUUUUAUAUAATT 914 UUAUAUAAAAUUUGAGCUUTT AD-14942 2743-2761 915CUGGACAUGGAUCAAGCACTT 916 GUGCUUGAUCCAUGUCCAGTT AD-14943 4155-4173 917UCAAAUUUUAUAUAAGAAATT 918 UUUCUUAUAUAAAAUUUGATT AD-14944 2871-2889 919UGUGCAGUGGAAAGAAAGGTT 920 CCUUUCUUUCCACUGCACATT AD-14945 4786-4804 921UUGGUACAUGGAAUAGUUCTT 922 GAACUAUUCCAUGUACCAATT AD-14946 4364-4382 923AAGUGGGAUGAAGACCUGUTT 924 ACAGGUCUUCAUCCCACUUTT AD-14947 4359-4377 925GGAUGAAGACCUGUUUUGCTT 926 GCAAAACAGGUCUUCAUCCTT AD-14948 2744-2762 927UCUGGACAUGGAUCAAGCATT 928 UGCUUGAUCCAUGUCCAGATT AD-14949 4787-4805 929UUUGGUACAUGGAAUAGUUTT 930 AACUAUUCCAUGUACCAAATT

Residual luciferase Relative activity siRNA activity (relative to SD of(normalized to  SD of Table la-2 control residual Residual positiverelative duplex siRNA luciferase luciferase control luc- siRNARelative siRNA name treated cells) activity activity +/− SD siRNA)activity activity +/− SD AD-14742 40.85 4.38 41 ± 4% 82.24 8.83 82 ± 9%AD-14743 20.92 4.10 21 ± 4% 109.97 21.56 110 ± 22% AD-14744 62.20 4.4762 ± 4% 52.56 3.78 53 ± 4% AD-14745 43.97 2.60 44 ± 3% 77.91 4.60 78 ±5% AD-14746 24.52 1.96 25 ± 2% 104.96 8.38 105 ± 8%  AD-14747 32.67 4.5133 ± 5% 93.62 12.94  94 ± 13% AD-14748 93.99 3.23 94 ± 3% 8.36 0.29  8 ±0% AD-14749 55.16 2.81 55 ± 3% 62.35 3.18 62 ± 3% AD-14750 30.86 3.1131 ± 3% 96.14 9.70  96 ± 10% AD-14751 54.44 4.03 54 ± 4% 63.35 4.69 63 ±5% AD-14752 53.88 7.58 54 ± 8% 64.13 9.02 64 ± 9% AD-14753 35.24 7.4535 ± 7% 90.05 19.03  90 ± 19% AD-14754 70.39 2.80 70 ± 3% 41.17 1.6441 ± 2% AD-14755 41.80 1.60 42 ± 2% 80.93 3.10 81 ± 3% AD-14756 56.693.05 57 ± 3% 60.22 3.24 60 ± 3% AD-14757 39.16 2.16 39 ± 2% 84.60 4.6785 ± 5% AD-14758 39.79 2.95 40 ± 3% 83.72 6.22 84 ± 6% AD-14759 30.621.01 31 ± 1% 96.48 3.20 96 ± 3% AD-14760 28.14 2.74 28 ± 3% 99.93 9.72100 ± 10% AD-14761 67.42 2.83 67 ± 3% 45.30 1.90 45 ± 2% AD-14762 36.101.30 36 ± 1% 88.85 3.21 89 ± 3% AD-14763 49.39 6.77 49 ± 7% 78.14 10.71 78 ± 11% AD-14764 74.04 5.32 74 ± 5% 40.09 2.88 40 ± 3% AD-14765 50.8410.47 51 ± 10% 75.91 15.63  76 ± 16% AD-14766 72.59 3.55 73 ± 4% 42.322.07 42 ± 2% AD-14767 34.82 7.41 35 ± 7% 100.63 21.40 101 ± 21% AD-1476848.68 6.31 49 ± 6% 79.24 10.27  79 ± 10% AD-14769 39.07 5.53 39 ± 6%94.08 13.31  94 ± 13% AD-14770 45.59 5.89 46 ± 6% 84.01 10.85  84 ± 11%AD-14771 45.57 4.10 46 ± 4% 84.04 7.56 84 ± 8% AD-14772 33.12 3.64 33 ±4% 103.26 11.36 103 ± 11% AD-14773 37.38 5.72 37 ± 6% 96.69 14.78  97 ±15% AD-14774 42.38 4.41 42 ± 4% 88.96 9.26 89 ± 9% AD-14775 46.59 3.0047 ± 3% 82.47 5.31 82 ± 5% AD-14776 71.28 8.67 71 ± 9% 44.35 5.40 44 ±5% AD-14777 64.55 6.21 65 ± 6% 54.74 5.26 55 ± 5% AD-14778 60.45 8.9160 ± 9% 61.07 9.00 61 ± 9% AD-14779 32.46 0.82 32 ± 1% 104.27 2.63 104 ±3%  AD-14780 22.96 2.86 23 ± 3% 118.94 14.81 119 ± 15% AD-14781 56.999.43 57 ± 9% 66.41 10.99  66 ± 11% AD-14782 29.90 8.74 30 ± 9% 108.2431.65 108 ± 32% AD-14783 42.63 6.57 43 ± 7% 88.58 13.66  89 ± 14%AD-14784 67.06 1.35 67 ± 1% 50.86 1.03 51 ± 1% AD-14785 48.90 3.32 49 ±3% 78.89 5.35 79 ± 5% AD-14786 27.74 2.06 28 ± 2% 111.57 8.29 112 ± 8% AD-14787 38.77 6.24 39 ± 6% 94.53 15.22  95 ± 15% AD-14788 32.84 8.6033 ± 9% 103.70 27.17 104 ± 27% AD-14789 46.96 1.70 47 ± 2% 81.89 2.9682 ± 3% AD-14790 43.61 4.90 44 ± 5% 87.06 9.79  87 ± 10% AD-14791 35.554.34 36 ± 4% 99.51 12.15 100 ± 12% AD-14792 38.22 3.51 38 ± 4% 95.388.75 95 ± 9% AD-14793 90.85 5.92 91 ± 6% 14.13 0.92 14 ± 1% AD-1479483.37 3.27 83 ± 3% 25.68 1.01 26 ± 1% AD-14795 55.06 3.61 55 ± 4% 69.384.55 69 ± 5% AD-14796 30.98 5.78 31 ± 6% 106.56 19.89 107 ± 20% AD-1479728.95 3.15 29 ± 3% 109.70 11.95 110 ± 12% AD-14798 67.39 3.70 67 ± 4%50.35 2.76 50 ± 3% AD-14799 66.83 4.72 67 ± 5% 51.21 3.61 51 ± 4%AD-14800 33.26 5.72 33 ± 6% 103.04 17.71 103 ± 18% AD-14801 39.15 4.5739 ± 5% 93.96 10.97  94 ± 11% AD-14802 91.20 5.35 91 ± 5% 13.58 0.8014 ± 1% AD-14803 34.15 7.94 34 ± 8% 101.67 23.64 102 ± 24% AD-1480430.08 6.54 30 ± 7% 107.96 23.48 108 ± 23% AD-14805 32.44 4.27 32 ± 4%104.31 13.73 104 ± 14% AD-14806 35.62 3.11 36 ± 3% 99.41 8.67 99 ± 9%AD-14807 28.27 7.28 28 ± 7% 110.76 28.52 111 ± 29% AD-14808 30.29 3.9630 ± 4% 107.63 14.08 108 ± 14% AD-14809 31.59 4.46 32 ± 4% 105.63 14.91106 ± 15% AD-14810 30.11 5.71 30 ± 6% 107.91 20.46 108 ± 20% AD-1481155.27 6.82 55 ± 7% 69.06 8.52 69 ± 9% AD-14812 45.27 5.99 45 ± 6% 84.5111.19  85 ± 11% AD-14813 77.97 7.01 78 ± 7% 34.01 3.06 34 ± 3% AD-1481429.54 3.56 30 ± 4% 108.78 13.09 109 ± 13% AD-14815 65.04 3.18 65 ± 3%53.97 2.64 54 ± 3% AD-14816 64.03 4.63 64 ± 5% 55.53 4.02 56 ± 4%AD-14817 37.83 2.89 38 ± 3% 95.99 7.33 96 ± 7% AD-14818 28.88 5.60 29 ±6% 109.82 21.30 110 ± 21% AD-14819 92.90 4.87 93 ± 5% 10.97 0.58 11 ± 1%AD-14820 75.41 3.69 75 ± 4% 37.97 1.86 38 ± 2% AD-14821 73.08 6.22 73 ±6% 41.57 3.54 42 ± 4% AD-14822 86.39 9.34 86 ± 9% 21.02 2.27 21 ± 2%AD-14823 96.50 10.46  97 ± 10% 5.40 0.59  5 ± 1% AD-14824 32.62 3.4133 ± 3% 104.03 10.89 104 ± 11% AD-14825 102.71 7.66 103 ± 8%  −4.18 0.31−4 ± 0% AD-14826 92.45 5.66 92 ± 6% 11.66 0.71 12 ± 1% AD-14827 63.4616.38  63 ± 16% 46.00 11.88  46 ± 12% AD-14828 45.99 15.21  46 ± 15%67.99 22.49  68 ± 22% AD-14829 40.54 16.03  41 ± 16% 74.86 29.60  75 ±30% AD-14830 117.10 3.66 117± 4%  −21.52 0.67 −22 ± 1%  AD-14831 54.7821.12  55 ± 21% 56.93 21.95  57 ± 22% AD-14832 67.07 10.81  67 ± 11%41.46 6.68 41 ± 7% AD-14833 71.52 11.90  72 ± 12% 35.85 5.97 36 ± 6%AD-14834 58.05 16.37  58 ± 16% 52.81 14.89  53 ± 15% AD-14835 93.36 5.4393 ± 5% 8.36 0.49  8 ± 0% AD-14836 108.84 4.85 109 ± 5%  −11.13 0.50−11 ± 0%  AD-14837 106.68 10.06 107 ± 10% −8.41 0.79 −8 ± 1% AD-1483837.06 6.68 37 ± 7% 79.23 14.28  79 ± 14% AD-14839 36.03 7.54 36 ± 8%80.53 16.84  81 ± 17% AD-14840 38.51 5.90 39 ± 6% 77.40 11.86  77 ± 12%AD-14841 110.86 8.91 111 ± 9% −13.67 1.10 −14 ± 1%  AD-14842 34.83 5.5135 ± 6% 82.04 12.98  82 ± 13% AD-14843 23.75 6.04 24 ± 6% 95.99 24.41 96 ± 24% AD-14844 27.47 5.29 27 ± 5% 91.30 17.57  91 ± 18% AD-1484593.12 4.70 93 ± 5% 8.67 0.44  9 ± 0% AD-14846 81.72 8.26 82 ± 8% 23.012.33 23 ± 2% AD-14847 77.89 5.29 78 ± 5% 27.83 1.89 28 ± 2% AD-1484844.40 4.95 44 ± 5% 69.99 7.81 70 ± 8% AD-14849 46.41 5.08 46 ± 5% 67.467.38 67 ± 7% AD-14850 35.52 6.70 36 ± 7% 81.17 15.31  81 ± 15% AD-1485136.07 1.13 36 ± 1% 102.63 3.22 103 ± 3%  AD-14852 67.98 6.75 68 ± 7%51.41 5.11 51 ± 5% AD-14853 69.44 3.07 69 ± 3% 49.05 2.17 49 ± 2%AD-14854 29.12 6.88 29 ± 7% 113.79 26.89 114 ± 27% AD-14855 36.04 7.0736 ± 7% 102.68 20.14 103 ± 20% AD-14856 33.61 7.93 34 ± 8% 106.57 25.15107 ± 25% AD-14857 50.76 8.76 51 ± 9% 79.04 13.64  79 ± 14% AD-1485853.60 7.26 54 ± 7% 74.49 10.09  74 ± 10% AD-14859 39.07 9.34 39 ± 9%97.82 23.38  98 ± 23% AD-14860 62.78 6.85 63 ± 7% 59.75 6.52 60 ± 7%AD-14861 87.47 1.86 87 ± 2% 20.12 0.43 20 ± 0% AD-14862 79.95 4.02 80 ±4% 32.19 1.62 32 ± 2% AD-14863 30.46 4.49 30 ± 4% 111.64 16.46 112 ± 16%AD-14864 33.18 5.07 33 ± 5% 107.26 16.38 107 ± 16% AD-14865 26.25 3.9826 ± 4% 118.39 17.96 118 ± 18% AD-14866 36.73 1.24 37 ± 1% 101.57 3.44102 ± 3%  AD-14867 33.16 3.13 33 ± 3% 107.30 10.12 107 ± 10% AD-1486829.91 4.56 30 ± 5% 112.52 17.16 113 ± 17% AD-14869 28.24 3.66 28 ± 4%115.20 14.91 115 ± 15% AD-14870 50.37 3.04 50 ± 3% 79.67 4.81 80 ± 5%AD-14871 39.37 5.11 39 ± 5% 97.32 12.63  97 ± 13% AD-14872 34.71 4.1235 ± 4% 104.82 12.43 105 ± 12% AD-14873 32.14 1.79 32 ± 2% 108.93 6.07109 ± 6%  AD-14874 101.77 4.87 102 ± 5%  −2.85 0.14 <3 ± 0% AD-1487580.81 4.39 81 ± 4% 30.80 1.67 31 ± 2% AD-14876 30.74 1.88 31 ± 2% 111.186.81 111 ± 7%  AD-14877 57.38 2.84 57 ± 3% 68.42 3.39 68 ± 3% AD-1487870.23 3.35 70 ± 3% 47.79 2.28 48 ± 2% AD-14879 79.03 7.72 79 ± 8% 33.663.29 34 ± 3% AD-14880 21.65 2.46 22 ± 2% 125.78 14.28 126 ± 14% AD-1488127.66 1.71 28 ± 2% 116.13 7.17 116 ± 7%  AD-14882 34.01 2.94 34 ± 3%105.93 9.16 106 ± 9%  AD-14883 40.62 3.22 41 ± 3% 95.33 7.56 95 ± 8%AD-14884 35.73 5.94 36 ± 6% 103.18 17.14 103 ± 17% AD-14885 47.40 7.6547 ± 8% 84.45 13.63  84 ± 14% AD-14886 37.23 3.94 37 ± 4% 100.76 10.67101 ± 11% AD-14887 42.94 7.26 43 ± 7% 91.61 15.50  92 ± 15% AD-1488832.58 4.06 33 ± 4% 108.24 13.50 108 ± 14% AD-14889 83.09 2.98 83 ± 3%27.15 0.97 27 ± 1% AD-14890 59.49 2.94 59 ± 3% 65.04 3.22 65 ± 3%AD-14891 21.93 5.52 22 ± 6% 125.32 31.52 125 ± 32% AD-14892 72.69 2.1973 ± 2% 43.84 1.32 44 ± 1% AD-14893 24.43 7.07 24 ± 7% 121.32 35.11121 ± 35% AD-14894 33.84 5.08 34 ± 5% 106.20 15.95 106 ± 16% AD-1489521.68 4.46 22 ± 4% 125.73 25.84 126 ± 26% AD-14896 26.99 5.01 27 ± 5%117.20 21.73 117 ± 22% AD-14897 29.04 2.72 29 ± 3% 113.92 10.67 114 ±11% AD-14898 32.64 4.87 33 ± 5% 108.14 16.13 108 ± 16% AD-14899 61.714.59 62 ± 5% 61.47 4.57 61 ± 5% AD-14900 31.01 2.84 31 ± 3% 110.75 10.14111 ± 10% AD-14901 31.47 1.57 31 ± 2% 110.01 5.49 110 ± 5%  AD-1490276.99 0.55 77 ± 1% 36.95 0.26 37 ± 0% AD-14903 20.55 3.55 21 ± 4% 127.5522.05 128 ± 22% AD-14904 22.65 6.87 23 ± 7% 124.18 37.68 124 ± 38%AD-14905 56.98 4.94 57 ± 5% 69.07 5.99 69 ± 6% AD-14906 34.20 3.66 34 ±4% 105.63 11.29 106 ± 11% AD-14907 28.59 8.12 29 ± 8% 114.64 32.56 115 ±33% AD-14908 34.08 3.36 34 ± 3% 105.82 10.44 106 ± 10% AD-14909 76.572.33 77 ± 2% 37.61 1.15 38 ± 1% AD-14910 46.50 4.14 46 ± 4% 85.89 7.6486 ± 8% AD-14911 29.62 2.02 30 ± 2% 112.99 7.69 113 ± 8%  AD-14912 22.270.48 22 ± 0% 124.78 2.69 125 ± 3%  AD-14913 59.80 2.85 60 ± 3% 64.533.08 65 ± 3% AD-14914 93.21 5.10 93 ± 5% 10.90 0.60 11 ± 1% AD-1491525.99 4.45 26 ± 4% 118.82 20.34 119 ± 20% AD-14916 48.20 1.46 48 ± 1%83.16 2.51 83 ± 3% AD-14917 41.03 3.07 41 ± 3% 94.67 7.08 95 ± 7%AD-14918 110.62 6.34 111 ± 6%  −17.04 0.98 −17 ± 1%  AD-14919 73.66 3.6874 ± 4% 42.29 2.11 42 ± 2% AD-14920 19.80 1.72 20 ± 2% 128.75 11.20129 ± 11% AD-14921 33.13 1.14 33 ± 1% 107.34 3.71 107 ± 4%  AD-1492252.94 6.99 53 ± 7% 63.41 8.37 63 ± 8% AD-14923 33.77 8.92 34 ± 9% 89.2323.56  89 ± 24% AD-14924 64.47 10.96  64 ± 11% 47.86 8.13 48 ± 8%AD-14925 97.16 7.57 97 ± 8% 3.83 0.30  4 ± 0% AD-14926 27.29 8.79 27 ±9% 97.96 31.56  98 ± 32% AD-14927 27.02 10.01  27 ± 10% 98.33 36.42 98 ± 36% AD-14928 76.75 4.78 77 ± 5% 31.32 1.95 31 ± 2% AD-14929 32.929.44 33 ± 9% 90.38 25.93  90 ± 26% AD-14930 31.00 9.40 31 ± 9% 92.9728.21  93 ± 28% AD-14931 31.36 8.73 31 ± 9% 92.48 25.74  92 ± 26%AD-14932 32.42 9.01 32 ± 9% 91.05 25.29  91 ± 25% AD-14933 39.94 6.9640 ± 7% 80.92 14.10  81 ± 14% AD-14934 42.94 7.66 43 ± 8% 76.88 13.71 77 ± 14% AD-14935 47.74 8.48 48 ± 8% 70.41 12.51  70 ± 13% AD-1493635.21 4.02 35 ± 4% 87.29 9.97  87 ± 10% AD-14937 89.25 3.53 89 ± 4%14.48 0.57 14 ± 1% AD-14938 29.38 6.46 29 ± 6% 95.15 20.91  95 ± 21%AD-14939 26.45 8.33 26 ± 8% 99.09 31.21  99 ± 31% AD-14940 77.50 6.5178 ± 7% 30.31 2.55 30 ± 3% AD-14941 36.40 10.76  36 ± 11% 85.68 25.32 86 ± 25% AD-14942 65.11 5.84 65 ± 6% 47.01 4.22 47 ± 4% AD-14943 89.964.69 90 ± 5% 13.52 0.71 14 ± 1% AD-14944 48.98 5.85 49 ± 6% 68.74 8.2169 ± 8% AD-14945 43.45 3.29 43 ± 3% 76.18 5.77 76 ± 6% AD-14946 41.251.26 41 ± 1% 79.15 2.43 79 ± 2% AD-14947 42.10 7.34 42 ± 7% 78.01 13.60 78 ± 14% AD-14948 42.39 5.75 42 ± 6% 77.62 10.54  78 ± 11% AD-1494927.68 5.79 28 ± 6% 97.44 20.39  97 ± 20%

Tables 1b-1 and 1b-2

TABLE 1b siRNAs targeting JCV transcripts for primaryscreen; 1b-1: sequences; 1b-2: assay results.C* column describes chemistries as follows: Table 1b-1 SEQ SEQ duplexposition in ID sense strand ID antisense strand name C* consensus NO:sequence (5′-3′) NO: sequence (5′-3′) AD-12598 a 1426-1444 1AcuuuuAGGGuuGuAcGGGTsT 2 CcCGuAcAACCCuAAAAGUTsT AD-12708 b 1426-1444 3AcuuuuAGGGuuGuAcGGGTsT 4 CCCGuAcAACCCuAAAAGUTsT AD-12599 a 1427-1445 5cuuuuAGGGuuGuAcGGGATsT 6 UcCCGuAcAACCCuAAAAGTsT AD-12709 b 1427-1445 7cuuuuAGGGuuGuAcGGGATsT 8 UCCCGuAcAACCCuAAAAGTsT AD-12600 a 2026-2044 9cAGAGcAcAAGGcGuAccuTsT 10 AgGuACGCCUUGUGCUCUGTsT AD-12710 b 2026-2044 11cAGAGcAcAAGGcGuAccuTsT 12 AGGuACGCCUUGUGCUCUGTsT AD-12784 c 2026-2044 13caGAGcAcAAGGcGuAccuTsT 14 AGGuACGCCUUGUGCUCUGTsT AD-12832 d 2026-2044 15caGAGcAcAAGGcGuAccuTsT 16 AgGuACGCCUUGUGCUCUGTsT AD-12601 a 1431-1449 17uAGGGuuGuAcGGGAcuGuTsT 18 AcAGUCCCGuAcAACCCuATsT AD-12785 c 1431-1449 19uaGGGuuGuAcGGGAcuGuTsT 20 AcAGUCCCGuAcAACCCuATsT AD-12602 a 1432-1450 21AGGGuuGuAcGGGAcuGuATsT 22 uacAGUCCCGuAcAACCCUTsT AD-12711 b 1432-1450 23AGGGuuGuAcGGGAcuGuATsT 24 uAcAGUCCCGuAcAACCCUTsT AD-12786 c 1432-1450 25AgGGuuGuAcGGGAcuGuATsT 26 uAcAGUCCCGuAcAACCCUTsT AD-12833 d 1432-1450 27AgGGuuGuAcGGGAcuGuATsT 28 uacAGUCCCGuAcAACCCUTsT AD-12603 a 1436-1454 29uuGuAcGGGAcuGuAAcAcTsT 30 GuGUuAcAGUCCCGuAcAATsT AD-12712 b 1436-1454 31uuGuAcGGGAcuGuAAcAcTsT 32 GUGUuAcAGUCCCGuAcAATsT AD-12604 a 4794-4812 33GccuGAuuuuGGuAcAuGGTsT 34 CcAUGuACcAAAAUcAGGCTsT AD-12605 a 5099-5117 35GAAGuAGuAAGGGcGuGGATsT 36 UccACGCCCUuACuACUUCTsT AD-12713 b 5099-5117 37GAAGuAGuAAGGGcGuGGATsT 38 UCcACGCCCUuACuACUUCTsT AD-12787 c 5099-5117 39GaAGuAGuAAGGGcGuGGATsT 40 UCcACGCCCUuACuACUUCTsT AD-12834 d 5099-5117 41GaAGuAGuAAGGGcGuGGATsT 42 UccACGCCCUuACuACUUCTsT AD-12606 a 713-731 43AuAGGccuuAcuccuGAAATsT 44 UuUcAGGAGuAAGGCCuAUTsT AD-12714 b 713-731 45AuAGGccuuAcuccuGAAATsT 46 UUUcAGGAGuAAGGCCuAUTsT AD-12607 a 3946-3964 47GAcAGccAuAuGcAGuAGuTsT 48 AcuACUGcAuAUGGCUGUCTsT AD-12715 b 3946-3964 49GAcAGccAuAuGcAGuAGuTsT 50 ACuACUGcAuAUGGCUGUCTsT AD-12788 c 3946-3964 51GacAGccAuAuGcAGuAGuTsT 52 ACuACUGcAuAUGGCUGUCTsT AD-12835 d 3946-3964 53GacAGccAuAuGcAGuAGuTsT 54 AcuACUGcAuAUGGCUGUCTsT AD-12608 a 1128-1146 55AAAcuAcuuGGGcAAuAGuTsT 56 AcuAUUGCCcAAGuAGUUUTsT AD-12716 b 1128-1146 57AAAcuAcuuGGGcAAuAGuTsT 58 ACuAUUGCCcAAGuAGUUUTsT AD-12789 c 1128-1146 59AaAcuAcuuGGGcAAuAGuTsT 60 ACuAUUGCCcAAGuAGUUUTsT AD-12836 d 1128-1146 61AaAcuAcuuGGGcAAuAGuTsT 62 AcuAUUGCCcAAGuAGUUUTsT AD-12609 a 525-543 63ucAGGuucAuGGGuGccGcTsT 64 GcGGcACCcAUGAACCUGATsT AD-12717 b 525-543 65ucAGGuucAuGGGuGccGcTsT 66 GCGGcACCcAUGAACCUGATsT AD-12610 a 5096-5114 67GuAGuAAGGGcGuGGAGGcTsT 68 GcCUCcACGCCCUuACuACTsT AD-12718 b 5096-5114 69GuAGuAAGGGcGuGGAGGcTsT 70 GCCUCcACGCCCUuACuACTsT AD-12611 a 4727-4745 71uAuuGcAAGGAAuGGccuATsT 72 uaGGCcAUUCCUUGcAAuATsT AD-12719 b 4727-4745 73uAuuGcAAGGAAuGGccuATsT 74 uAGGCcAUUCCUUGcAAuATsT AD-12790 c 4727-4745 75uauuGcAAGGAAuGGccuATsT 76 uAGGCcAUUCCUUGcAAuATsT AD-12837 d 4727-4745 77uauuGcAAGGAAuGGccuATsT 78 uaGGCcAUUCCUUGcAAuATsT AD-12612 a 5097-5115 79AGuAGuAAGGGcGuGGAGGTsT 80 CcUCcACGCCCUuACuACUTsT AD-12720 b 5097-5115 81AGuAGuAAGGGcGuGGAGGTsT 82 CCUCcACGCCCUuACuACUTsT AD-12791 c 5097-5115 83AguAGuAAGGGcGuGGAGGTsT 84 CCUCcACGCCCUuACuACUTsT AD-12838 d 5097-5115 85AguAGuAAGGGcGuGGAGGTsT 86 CcUCcACGCCCUuACuACUTsT AD-12613 a 4601-4619 87uGcuAuuGcuuuGAuuGcuTsT 88 AgcAAUcAAAGcAAuAGcATsT AD-12721 b 4601-4619 89uGcuAuuGcuuuGAuuGcuTsT 90 AGcAAUcAAAGcAAuAGcATsT AD-12792 c 4601-4619 91ugcuAuuGcuuuGAuuGcuTsT 92 AGcAAUcAAAGcAAuAGcATsT AD-12839 d 4601-4619 93ugcuAuuGcuuuGAuuGcuTsT 94 AgcAAUcAAAGcAAuAGcATsT AD-12614 a 4600-4618 95GcuAuuGcuuuGAuuGcuuTsT 96 AaGcAAUcAAAGcAAuAGCTsT AD-12722 b 4600-4618 97GcuAuuGcuuuGAuuGcuuTsT 98 AAGcAAUcAAAGcAAuAGCTsT AD-12615 a 1421-1439 99ccuuuAcuuuuAGGGuuGuTsT 100 AcAACCCuAAAAGuAAAGGTsT AD-12616 a 1424-1442101 uuAcuuuuAGGGuuGuAcGTsT 102 CguAcAACCCuAAAAGuAATsT AD-12723 b1424-1442 103 uuAcuuuuAGGGuuGuAcGTsT 104 CGuAcAACCCuAAAAGuAATsT AD-12617a 1403-1421 105 GcuccucAAuGGAuGuuGcTsT 106 GcAAcAUCcAUUGAGGAGCTsTAD-12618 a 1534-1552 107 uuAuAAGAGGAGGAGuAGATsT 108UcuACUCCUCCUCUuAuAATsT AD-12724 b 1534-1552 109 uuAuAAGAGGAGGAGuAGATsT110 UCuACUCCUCCUCUuAuAATsT AD-12619 a 5098-5116 111AAGuAGuAAGGGcGuGGAGTsT 112 CuCcACGCCCUuACuACUUTsT AD-12725 b 5098-5116113 AAGuAGuAAGGGcGuGGAGTsT 114 CUCcACGCCCUuACuACUUTsT AD-12793 c5098-5116 115 AaGuAGuAAGGGcGuGGAGTsT 116 CUCcACGCCCUuACuACUUTsT AD-12840d 5098-5116 117 AaGuAGuAAGGGcGuGGAGTsT 118 CuCcACGCCCUuACuACUUTsTAD-12620 a 1430-1448 119 uuAGGGuuGuAcGGGAcuGTsT 120caGUCCCGuAcAACCCuAATsT AD-12726 b 1430-1448 121 uuAGGGuuGuAcGGGAcuGTsT122 cAGUCCCGuAcAACCCuAATsT AD-12621 a 1701-1719 123GAcAuGcuuccuuGuuAcATsT 124 UguAAcAAGGAAGcAUGUCTsT AD-12727 b 1701-1719125 GAcAuGcuuccuuGuuAcATsT 126 UGuAAcAAGGAAGcAUGUCTsT AD-12794 c1701-1719 127 GacAuGcuuccuuGuuAcATsT 128 UGuAAcAAGGAAGcAUGUCTsT AD-12841d 1701-1719 129 GacAuGcuuccuuGuuAcATsT 130 UguAAcAAGGAAGcAUGUCTsTAD-12622 a 2066-2084 131 uGuuGAAuGuuGGGuuccuTsT 132AgGAACCcAAcAUUcAAcATsT AD-12728 b 2066-2084 133 uGuuGAAuGuuGGGuuccuTsT134 AGGAACCcAAcAUUcAAcATsT AD-12795 c 2066-2084 135uguuGAAuGuuGGGuuccuTsT 136 AGGAACCcAAcAUUcAAcATsT AD-12842 d 2066-2084137 uguuGAAuGuuGGGuuccuTsT 138 AgGAACCcAAcAUUcAAcATsT AD-12623 a4561-4579 139 AcuuAAcccAAGAAGcucuTsT 140 AgAGCUUCUUGGGUuAAGUTsT AD-12729b 4561-4579 141 AcuuAAcccAAGAAGcucuTsT 142 AGAGCUUCUUGGGUuAAGUTsTAD-12624 a 4797-4815 143 ucAGccuGAuuuuGGuAcATsT 144UguACcAAAAUcAGGCUGATsT AD-12730 b 4797-4815 145 ucAGccuGAuuuuGGuAcATsT146 UGuACcAAAAUcAGGCUGATsT AD-12625 a 1428-1446 147uuuuAGGGuuGuAcGGGAcTsT 148 GuCCCGuAcAACCCuAAAATsT AD-12731 b 1428-1446149 uuuuAGGGuuGuAcGGGAcTsT 150 GUCCCGuAcAACCCuAAAATsT AD-12626 a1429-1447 151 uuuAGGGuuGuAcGGGAcuTsT 152 AgUCCCGuAcAACCCuAAATsT AD-12732b 1429-1447 153 uuuAGGGuuGuAcGGGAcuTsT 154 AGUCCCGuAcAACCCuAAATsTAD-12627 a 662-680 155 ucccuuGcuAcuGuAGAGGTsT 156 CcUCuAcAGuAGcAAGGGATsTAD-12733 b 662-680 157 ucccuuGcuAcuGuAGAGGTsT 158 CCUCuAcAGuAGcAAGGGATsTAD-12628 a 663-681 159 cccuuGcuAcuGuAGAGGGTsT 160 CcCUCuAcAGuAGcAAGGGTsTAD-12734 b 663-681 161 cccuuGcuAcuGuAGAGGGTsT 162 CCCUCuAcAGuAGcAAGGGTsTAD-12629 a 1402-1420 163 uGcuccucAAuGGAuGuuGTsT 164caAcAUCcAUUGAGGAGcATsT AD-12735 b 1402-1420 165 uGcuccucAAuGGAuGuuGTsT166 cAAcAUCcAUUGAGGAGcATsT AD-12796 c 1402-1420 167ugcuccucAAuGGAuGuuGTsT 168 cAAcAUCcAUUGAGGAGcATsT AD-12843 d 1402-1420169 ugcuccucAAuGGAuGuuGTsT 170 caAcAUCcAUUGAGGAGcATsT AD-12630 a1398-1416 171 GAucuGcuccucAAuGGAuTsT 172 AuCcAUUGAGGAGcAGAUCTsT AD-12736b 1398-1416 173 GAucuGcuccucAAuGGAuTsT 174 AUCcAUUGAGGAGcAGAUCTsTAD-12797 c 1398-1416 175 GaucuGcuccucAAuGGAuTsT 176AUCcAUUGAGGAGcAGAUCTsT AD-12844 d 1398-1416 177 GaucuGcuccucAAuGGAuTsT178 AuCcAUUGAGGAGcAGAUCTsT AD-12631 a 1399-1417 179AucuGcuccucAAuGGAuGTsT 180 caUCcAUUGAGGAGcAGAUTsT AD-12737 b 1399-1417181 AucuGcuccucAAuGGAuGTsT 182 cAUCcAUUGAGGAGcAGAUTsT AD-12632 a1400-1418 183 ucuGcuccucAAuGGAuGuTsT 184 AcAUCcAUUGAGGAGcAGATsT AD-12633a 1401-1419 185 cuGcuccucAAuGGAuGuuTsT 186 AacAUCcAUUGAGGAGcAGTsTAD-12738 b 1401-1419 187 cuGcuccucAAuGGAuGuuTsT 188AAcAUCcAUUGAGGAGcAGTsT AD-12634 a 1435-1453 189 GuuGuAcGGGAcuGuAAcATsT190 UgUuAcAGUCCCGuAcAACTsT AD-12739 b 1435-1453 191GuuGuAcGGGAcuGuAAcATsT 192 UGUuAcAGUCCCGuAcAACTsT AD-12635 a 1437-1455193 uGuAcGGGAcuGuAAcAccTsT 194 GgUGUuAcAGUCCCGuAcATsT AD-12740 b1437-1455 195 uGuAcGGGAcuGuAAcAccTsT 196 GGUGUuAcAGUCCCGuAcATsT AD-12798c 1437-1455 197 uguAcGGGAcuGuAAcAccTsT 198 GGUGUuAcAGUCCCGuAcATsTAD-12845 d 1437-1455 199 uguAcGGGAcuGuAAcAccTsT 200GgUGUuAcAGUCCCGuAcATsT AD-12636 a 1438-1456 201 GuAcGGGAcuGuAAcAccuTsT202 AgGUGUuAcAGUCCCGuACTsT AD-12741 b 1438-1456 203GuAcGGGAcuGuAAcAccuTsT 204 AGGUGUuAcAGUCCCGuACTsT AD-12637 a 4796-4814205 cAGccuGAuuuuGGuAcAuTsT 206 AuGuACcAAAAUcAGGCUGTsT AD-12742 b4796-4814 207 cAGccuGAuuuuGGuAcAuTsT 208 AUGuACcAAAAUcAGGCUGTsT AD-12799c 4796-4814 209 caGccuGAuuuuGGuAcAuTsT 210 AUGuACcAAAAUcAGGCUGTsTAD-12846 d 4796-4814 211 caGccuGAuuuuGGuAcAuTsT 212AuGuACcAAAAUcAGGCUGTsT AD-12638 a 4992-5010 213 GAAuAGGGAGGAAuccAuGTsT214 caUGGAUUCCUCCCuAUUCTsT AD-12743 b 4992-5010 215GAAuAGGGAGGAAuccAuGTsT 216 cAUGGAUUCCUCCCuAUUCTsT AD-12800 c 4992-5010217 GaAuAGGGAGGAAuccAuGTsT 218 cAUGGAUUCCUCCCuAUUCTsT AD-12847 d4992-5010 219 GaAuAGGGAGGAAuccAuGTsT 220 caUGGAUUCCUCCCuAUUCTsT AD-12639a 4999-5017 221 AAGuGcuGAAuAGGGAGGATsT 222 UcCUCCCuAUUcAGcACUUTsTAD-12744 b 4999-5017 223 AAGuGcuGAAuAGGGAGGATsT 224UCCUCCCuAUUcAGcACUUTsT AD-12801 c 4999-5017 225 AaGuGcuGAAuAGGGAGGATsT226 UCCUCCCuAUUcAGcACUUTsT AD-12848 d 4999-5017 227AaGuGcuGAAuAGGGAGGATsT 228 UcCUCCCuAUUcAGcACUUTsT AD-12640 a 630-648 229AGGcuGcuGcuAcuAuAGATsT 230 UcuAuAGuAGcAGcAGCCUTsT AD-12745 b 630-648 231AGGcuGcuGcuAcuAuAGATsT 232 UCuAuAGuAGcAGcAGCCUTsT AD-12802 c 630-648 233AgGcuGcuGcuAcuAuAGATsT 234 UCuAuAGuAGcAGcAGCCUTsT AD-12849 d 630-648 235AgGcuGcuGcuAcuAuAGATsT 236 UcuAuAGuAGcAGcAGCCUTsT AD-12641 a 3947-3965237 AGAcAGccAuAuGcAGuAGTsT 238 CuACUGcAuAUGGCUGUCUTsT AD-12803 c3947-3965 239 AgAcAGccAuAuGcAGuAGTsT 240 CuACUGcAuAUGGCUGUCUTsT AD-12642a 524-542 241 uucAGGuucAuGGGuGccGTsT 242 CgGcACCcAUGAACCUGAATsT AD-12746b 524-542 243 uucAGGuucAuGGGuGccGTsT 244 CGGcACCcAUGAACCUGAATsT AD-12643a 3948-3966 245 uAGAcAGccAuAuGcAGuATsT 246 uaCUGcAuAUGGCUGUCuATsTAD-12747 b 3948-3966 247 uAGAcAGccAuAuGcAGuATsT 248uACUGcAuAUGGCUGUCuATsT AD-12804 c 3948-3966 249 uaGAcAGccAuAuGcAGuATsT250 uACUGcAuAUGGCUGUCuATsT AD-12850 d 3948-3966 251uaGAcAGccAuAuGcAGuATsT 252 uaCUGcAuAUGGCUGUCuATsT AD-12644 a 3900-3918253 GGAGcAuGAcuuuAAcccATsT 254 UgGGUuAAAGUcAUGCUCCTsT AD-12748 b3900-3918 255 GGAGcAuGAcuuuAAcccATsT 256 UGGGUuAAAGUcAUGCUCCTsT AD-12805c 3900-3918 257 GgAGcAuGAcuuuAAcccATsT 258 UGGGUuAAAGUcAUGCUCCTsTAD-12851 d 3900-3918 259 GgAGcAuGAcuuuAAcccATsT 260UgGGUuAAAGUcAUGCUCCTsT AD-12645 a 1417-1435 261 GuuGccuuuAcuuuuAGGGTsT262 CcCuAAAAGuAAAGGcAACTsT AD-12749 b 1417-1435 263GuuGccuuuAcuuuuAGGGTsT 264 CCCuAAAAGuAAAGGcAACTsT AD-12646 a 4565-4583265 uGuGAcuuAAcccAAGAAGTsT 266 CuUCUUGGGUuAAGUcAcATsT AD-12750 b4565-4583 267 uGuGAcuuAAcccAAGAAGTsT 268 CUUCUUGGGUuAAGUcAcATsT AD-12806c 4565-4583 269 uguGAcuuAAcccAAGAAGTsT 270 CUUCUUGGGUuAAGUcAcATsTAD-12852 d 4565-4583 271 uguGAcuuAAcccAAGAAGTsT 272CuUCUUGGGUuAAGUcAcATsT AD-12647 a 4598-4616 273 uAuuGcuuuGAuuGcuucATsT274 UgAAGcAAUcAAAGcAAuATsT AD-12751 b 4598-4616 275uAuuGcuuuGAuuGcuucATsT 276 UGAAGcAAUcAAAGcAAuATsT AD-12807 c 4598-4616277 uauuGcuuuGAuuGcuucATsT 278 UGAAGcAAUcAAAGcAAuATsT AD-12853 d4598-4616 279 uauuGcuuuGAuuGcuucATsT 280 UgAAGcAAUcAAAGcAAuATsT AD-12648a 2060-2078 281 AuAuccuGuuGAAuGuuGGTsT 282 CcAAcAUUcAAcAGGAuAUTsTAD-12649 a 4729-4747 283 uuuAuuGcAAGGAAuGGccTsT 284GgCcAUUCCUUGcAAuAAATsT AD-12752 b 4729-4747 285 uuuAuuGcAAGGAAuGGccTsT286 GGCcAUUCCUUGcAAuAAATsT AD-12650 a 1122-1140 287uGGAAGAAAcuAcuuGGGcTsT 288 GcCcAAGuAGUUUCUUCcATsT AD-12753 b 1122-1140289 uGGAAGAAAcuAcuuGGGcTsT 290 GCCcAAGuAGUUUCUUCcATsT AD-12808 c1122-1140 291 ugGAAGAAAcuAcuuGGGcTsT 292 GCCcAAGuAGUUUCUUCcATsT AD-12854d 1122-1140 293 ugGAAGAAAcuAcuuGGGcTsT 294 GcCcAAGuAGUUUCUUCcATsTAD-12651 a 4261-4279 295 AAGAcccuAAAGAcuuuccTsT 296GgAAAGUCUUuAGGGUCUUTsT AD-12754 b 4261-4279 297 AAGAcccuAAAGAcuuuccTsT298 GGAAAGUCUUuAGGGUCUUTsT AD-12809 c 4261-4279 299AaGAcccuAAAGAcuuuccTsT 300 GGAAAGUCUUuAGGGUCUUTsT AD-12855 d 4261-4279301 AaGAcccuAAAGAcuuuccTsT 302 GgAAAGUCUUuAGGGUCUUTsT AD-12652 a1412-1430 303 uGGAuGuuGccuuuAcuuuTsT 304 AaAGuAAAGGcAAcAUCcATsT AD-12755b 1412-1430 305 uGGAuGuuGccuuuAcuuuTsT 306 AAAGuAAAGGcAAcAUCcATsTAD-12810 c 1412-1430 307 ugGAuGuuGccuuuAcuuuTsT 308AAAGuAAAGGcAAcAUCcATsT AD-12856 d 1412-1430 309 ugGAuGuuGccuuuAcuuuTsT310 AaAGuAAAGGcAAcAUCcATsT AD-12653 a 4592-4610 311uuuGAuuGcuucAGAcAAuTsT 312 AuUGUCUGAAGcAAUcAAATsT AD-12756 b 4592-4610313 uuuGAuuGcuucAGAcAAuTsT 314 AUUGUCUGAAGcAAUcAAATsT AD-12654 a4991-5009 315 AAuAGGGAGGAAuccAuGGTsT 316 CcAUGGAUUCCUCCCuAUUTsT AD-12811c 4991-5009 317 AauAGGGAGGAAuccAuGGTsT 318 CcAUGGAUUCCUCCCuAUUTsTAD-12655 a 5004-5022 319 GGAcAAAGuGcuGAAuAGGTsT 320CcuAUUcAGcACUUUGUCCTsT AD-12757 b 5004-5022 321 GGAcAAAGuGcuGAAuAGGTsT322 CCuAUUcAGcACUUUGUCCTsT AD-12812 c 5004-5022 323GgAcAAAGuGcuGAAuAGGTsT 324 CCuAUUcAGcACUUUGUCCTsT AD-12857 d 5004-5022325 GgAcAAAGuGcuGAAuAGGTsT 326 CcuAUUcAGcACUUUGUCCTsT AD-12656 a5005-5023 327 uGGAcAAAGuGcuGAAuAGTsT 328 CuAUUcAGcACUUUGUCcATsT AD-12813c 5005-5023 329 ugGAcAAAGuGcuGAAuAGTsT 330 CuAUUcAGcACUUUGUCcATsTAD-12657 a 654-672 331 AAAuuGcAucccuuGcuAcTsT 332 GuAGcAAGGGAUGcAAUUUTsTAD-12814 c 654-672 333 AaAuuGcAucccuuGcuAcTsT 334 GuAGcAAGGGAUGcAAUUUTsTAD-12658 a 659-677 335 GcAucccuuGcuAcuGuAGTsT 336 CuAcAGuAGcAAGGGAUGCTsTAD-12659 a 4273-4291 337 AAAAAAAGGuAGAAGAcccTsT 338GgGUCUUCuACCUUUUUUUTsT AD-12758 b 4273-4291 339 AAAAAAAGGuAGAAGAcccTsT340 GGGUCUUCuACCUUUUUUUTsT AD-12815 c 4273-4291 341AaAAAAAGGuAGAAGAcccTsT 342 GGGUCUUCuACCUUUUUUUTsT AD-12858 d 4273-4291343 AaAAAAAGGuAGAAGAcccTsT 344 GgGUCUUCuACCUUUUUUUTsT AD-12660 a2025-2043 345 AcAGAGcAcAAGGcGuAccTsT 346 GguACGCCUUGUGCUCUGUTsT AD-12759b 2025-2043 347 AcAGAGcAcAAGGcGuAccTsT 348 GGuACGCCUUGUGCUCUGUTsTAD-12661 a 4791-4809 349 uGAuuuuGGuAcAuGGAAuTsT 350AuUCcAUGuACcAAAAUcATsT AD-12760 b 4791-4809 351 uGAuuuuGGuAcAuGGAAuTsT352 AUUCcAUGuACcAAAAUcATsT AD-12816 c 4791-4809 353ugAuuuuGGuAcAuGGAAuTsT 354 AUUCcAUGuACcAAAAUcATsT AD-12859 d 4791-4809355 ugAuuuuGGuAcAuGGAAuTsT 356 AuUCcAUGuACcAAAAUcATsT AD-12662 a1433-1451 357 GGGuuGuAcGGGAcuGuAATsT 358 UuAcAGUCCCGuAcAACCCTsT AD-12817c 1433-1451 359 GgGuuGuAcGGGAcuGuAATsT 360 UuAcAGUCCCGuAcAACCCTsTAD-12663 a 1434-1452 361 GGuuGuAcGGGAcuGuAAcTsT 362GuuAcAGUCCCGuAcAACCTsT AD-12761 b 1434-1452 363 GGuuGuAcGGGAcuGuAAcTsT364 GUuAcAGUCCCGuAcAACCTsT AD-12818 c 1434-1452 365GguuGuAcGGGAcuGuAAcTsT 366 GUuAcAGUCCCGuAcAACCTsT AD-12860 d 1434-1452367 GguuGuAcGGGAcuGuAAcTsT 368 GuuAcAGUCCCGuAcAACCTsT AD-12664 a1440-1458 369 AcGGGAcuGuAAcAccuGcTsT 370 GcAGGUGUuAcAGUCCCGUTsT AD-12665a 1442-1460 371 GGGAcuGuAAcAccuGcucTsT 372 GaGcAGGUGUuAcAGUCCCTsTAD-12762 b 1442-1460 373 GGGAcuGuAAcAccuGcucTsT 374GAGcAGGUGUuAcAGUCCCTsT AD-12819 c 1442-1460 375 GgGAcuGuAAcAccuGcucTsT376 GAGcAGGUGUuAcAGUCCCTsT AD-12861 d 1442-1460 377GgGAcuGuAAcAccuGcucTsT 378 GaGcAGGUGUuAcAGUCCCTsT AD-12666 a 1608-1626379 AcuccAGAAAuGGGuGAccTsT 380 GgUcACCcAUUUCUGGAGUTsT AD-12763 b1608-1626 381 AcuccAGAAAuGGGuGAccTsT 382 GGUcACCcAUUUCUGGAGUTsT AD-12667a 4793-4811 383 ccuGAuuuuGGuAcAuGGATsT 384 UccAUGuACcAAAAUcAGGTsTAD-12764 b 4793-4811 385 ccuGAuuuuGGuAcAuGGATsT 386UCcAUGuACcAAAAUcAGGTsT AD-12668 a 5001-5019 387 cAAAGuGcuGAAuAGGGAGTsT388 CuCCCuAUUcAGcACUUUGTsT AD-12765 b 5001-5019 389cAAAGuGcuGAAuAGGGAGTsT 390 CUCCCuAUUcAGcACUUUGTsT AD-12820 c 5001-5019391 caAAGuGcuGAAuAGGGAGTsT 392 CUCCCuAUUcAGcACUUUGTsT AD-12862 d5001-5019 393 caAAGuGcuGAAuAGGGAGTsT 394 CuCCCuAUUcAGcACUUUGTsT AD-12669a 5066-5084 395 ccAGGGAAAuucccuuGuuTsT 396 AacAAGGGAAUUUCCCUGGTsTAD-12766 b 5066-5084 397 ccAGGGAAAuucccuuGuuTsT 398AAcAAGGGAAUUUCCCUGGTsT AD-12670 a 5069-5087 399 AGGccAGGGAAAuucccuuTsT400 AaGGGAAUUUCCCUGGCCUTsT AD-12767 b 5069-5087 401AGGccAGGGAAAuucccuuTsT 402 AAGGGAAUUUCCCUGGCCUTsT AD-12821 c 5069-5087403 AgGccAGGGAAAuucccuuTsT 404 AAGGGAAUUUCCCUGGCCUTsT AD-12863 d5069-5087 405 AgGccAGGGAAAuucccuuTsT 406 AaGGGAAUUUCCCUGGCCUTsT AD-12671a 564-582 407 uAGuuGcuAcuGuuucuGATsT 408 UcAGAAAcAGuAGcAACuATsT AD-12822c 564-582 409 uaGuuGcuAcuGuuucuGATsT 410 UcAGAAAcAGuAGcAACuATsT AD-12672a 633-651 411 cuGcuGcuAcuAuAGAAGuTsT 412 AcUUCuAuAGuAGcAGcAGTsT AD-12768b 633-651 413 cuGcuGcuAcuAuAGAAGuTsT 414 ACUUCuAuAGuAGcAGcAGTsT AD-12673a 634-652 415 uGcuGcuAcuAuAGAAGuuTsT 416 AaCUUCuAuAGuAGcAGcATsT AD-12769b 634-652 417 uGcuGcuAcuAuAGAAGuuTsT 418 AACUUCuAuAGuAGcAGcATsT AD-12823c 634-652 419 ugcuGcuAcuAuAGAAGuuTsT 420 AACUUCuAuAGuAGcAGcATsT AD-12864d 634-652 421 ugcuGcuAcuAuAGAAGuuTsT 422 AaCUUCuAuAGuAGcAGcATsT AD-12674a 635-653 423 GcuGcuAcuAuAGAAGuuGTsT 424 caACUUCuAuAGuAGcAGCTsT AD-12770b 635-653 425 GcuGcuAcuAuAGAAGuuGTsT 426 cAACUUCuAuAGuAGcAGCTsT AD-12675a 636-654 427 cuGcuAcuAuAGAAGuuGATsT 428 UcAACUUCuAuAGuAGcAGTsT AD-12676a 637-655 429 uGcuAcuAuAGAAGuuGAATsT 430 UucAACUUCuAuAGuAGcATsT AD-12771b 637-655 431 uGcuAcuAuAGAAGuuGAATsT 432 UUcAACUUCuAuAGuAGcATsT AD-12824c 637-655 433 ugcuAcuAuAGAAGuuGAATsT 434 UUcAACUUCuAuAGuAGcATsT AD-12865d 637-655 435 ugcuAcuAuAGAAGuuGAATsT 436 UucAACUUCuAuAGuAGcATsT AD-12677a 912-930 437 cAGAAGAcuAcuAuGAuAuTsT 438 AuAUcAuAGuAGUCUUCUGTsT AD-12825c 912-930 439 caGAAGAcuAcuAuGAuAuTsT 440 AuAUcAuAGuAGUCUUCUGTsT AD-12678a 4153-4171 441 AAAuuuuAuAuAAGAAAcuTsT 442 AgUUUCUuAuAuAAAAUUUTsTAD-12772 b 4153-4171 443 AAAuuuuAuAuAAGAAAcuTsT 444AGUUUCU uAuAuAAAAUUUTsT AD-12826 c 4153-4171 445 AaAuuuuAuAuAAGAAAcuTsT446 AGUUUCU uAuAuAAAAUUUTsT AD-12866 d 4153-4171 447AaAuuuuAuAuAAGAAAcuTsT 448 AgUUUCUuAuAuAAAAUUUTsT AD-12679 a 4779-4797449 AuGGAAuAGuucAGAGGuuTsT 450 AaCCUCUGAACuAUUCcAUTsT AD-12773 b4779-4797 451 AuGGAAuAGuucAGAGGuuTsT 452 AACCUCUGAACuAUUCcAUTsT AD-12680a 4780-4798 453 cAuGGAAuAGuucAGAGGuTsT 454 AcCUCUGAACuAUUCcAUGTsTAD-12774 b 4780-4798 455 cAuGGAAuAGuucAGAGGuTsT 456ACCUCUGAACuAUUCcAUGTsT AD-12827 c 4780-4798 457 cauGGAAuAGuucAGAGGuTsT458 ACCUCUGAACuAUUCcAUGTsT AD-12867 d 4780-4798 459cauGGAAuAGuucAGAGGuTsT 460 AcCUCUGAACuAUUCcAUGTsT AD-12681 a 4781-4799461 AcAuGGAAuAGuucAGAGGTsT 462 CcUCUGAACuAUUCcAUGUTsT AD-12775 b4781-4799 463 AcAuGGAAuAGuucAGAGGTsT 464 CCUCUGAACuAUUCcAUGUTsT AD-12682a 4784-4802 465 GGuAcAuGGAAuAGuucAGTsT 466 CuGAACuAUUCcAUGuACCTsTAD-12776 b 4784-4802 467 GGuAcAuGGAAuAGuucAGTsT 468CUGAACuAUUCcAUGuACCTsT AD-12828 c 4784-4802 469 GguAcAuGGAAuAGuucAGTsT470 CUGAACuAUUCcAUGuACCTsT AD-12868 d 4784-4802 471GguAcAuGGAAuAGuucAGTsT 472 CuGAACuAUUCcAUGuACCTsT AD-12683 a 4785-4803473 uGGuAcAuGGAAuAGuucATsT 474 UgAACuAUUCcAUGuACcATsT AD-12777 b4785-4803 475 uGGuAcAuGGAAuAGuucATsT 476 UGAACuAUUCcAUGuACcATsT AD-12829c 4785-4803 477 ugGuAcAuGGAAuAGuucATsT 478 UGAACuAUUCcAUGuACcATsTAD-12869 d 4785-4803 479 ugGuAcAuGGAAuAGuucATsT 480UgAACuAUUCcAUGuACcATsT AD-12684 a 719-737 481 cuuAcuccuGAAAcAuAuGTsT 482cauAUGUUUcAGGAGuAAGTsT AD-12778 b 719-737 483 cuuAcuccuGAAAcAuAuGTsT 484cAuAUGUUUcAGGAGuAAGTsT AD-12685 a 909-927 485 AuccAGAAGAcuAcuAuGATsT 486UcAuAGuAGUCUUCUGGAUTsT AD-12686 a 1119-1137 487 uuuuGGAAGAAAcuAcuuGTsT488 caAGuAGUUUCUUCcAAAATsT AD-12779 b 1119-1137 489uuuuGGAAGAAAcuAcuuGTsT 490 cAAGuAGUUUCUUCcAAAATsT AD-12687 a 1121-1139491 uuGGAAGAAAcuAcuuGGGTsT 492 CccAAGuAGUUUCUUCcAATsT AD-12780 b1121-1139 493 uuGGAAGAAAcuAcuuGGGTsT 494 CCcAAGuAGUUUCUUCcAATsT AD-12688a 4357-4375 495 AuGAAGAccuGuuuuGccATsT 496 UgGcAAAAcAGGUCUUcAUTsTAD-12781 b 4357-4375 497 AuGAAGAccuGuuuuGccATsT 498UGGcAAAAcAGGUCUUcAUTsT AD-12689 a 4358-4376 499 GAuGAAGAccuGuuuuGccTsT500 GgcAAAAcAGGUCUUcAUCTsT AD-12782 b 4358-4376 501GAuGAAGAccuGuuuuGccTsT 502 GGcAAAAcAGGUCUUcAUCTsT AD-12830 c 4358-4376503 GauGAAGAccuGuuuuGccTsT 504 GGcAAAAcAGGUCUUcAUCTsT AD-12870 d4358-4376 505 GauGAAGAccuGuuuuGccTsT 506 GgcAAAAcAGGUCUUcAUCTsT AD-12690a 4360-4378 507 GGGAuGAAGAccuGuuuuGTsT 508 caAAAcAGGUCUUcAUCCCTsTAD-12783 b 4360-4378 509 GGGAuGAAGAccuGuuuuGTsT 510cAAAAcAGGUCUUcAUCCCTsT AD-12831 c 4360-4378 511 GgGAuGAAGAccuGuuuuGTsT512 cAAAAcAGGUCUUcAUCCCTsT AD-12871 d 4360-4378 513GgGAuGAAGAccuGuuuuGTsT 514 caAAAcAGGUCUUcAUCCCTsTDescription of chemistries:  a exo/endo-light +2′-0-methyl in position 2 of antisense b exo/endo-light: sense strand: dTsdT + 2′0Me@all Py; antisense strand: dTsdT + 2′0Me@ Py in uA,cA  c exo/endo-light +2′-0-methyl in position 2 of sense  d exo/endo-light +2′-0-methyl in position 2 of sense and antisense 

TABLE 1b-2 Residual luciferase SD of Relative siRNA SD of activity(relative to residual Residual activity (normalized relative Relativeduplex control siRNA luciferase luciferase to positive control siRNAsiRNA name treated cells) activity activity +/− SD luc-siRNA) activityactivity +/− SD AD-12598 91 11 91 ± 11%  9 2 9 ± 2% AD-12708 32 5 32 ±5% 76 17 76 ± 17% AD-12599 25 6 25 ± 6% 79 13 79 ± 13% AD-12709 16 4 16± 4% 97 26 97 ± 26% AD-12600 79 9 79 ± 9% 21 3 21 ± 3% AD-12710 25 4 25± 4% 85 24 85 ± 24% AD-12784 23 2 23 ± 2% 87 14 87 ± 14% AD-12832 84 1184 ± 11%  18 4 18 ± 4% AD-12601 102 8 102 ± 8%  −6 1 −6 ± 1% AD-12785 9510 95 ± 10%  6 1 6 ± 1% AD-12602 107 9 107 ± 9%  −11 2 −11 ± 2% AD-1271170 4 70 ± 4% 34 3 34 ± 3% AD-12786 69 8 69 ± 8% 35 7 35 ± 7% AD-12833 948 94 ± 8% 7 1 7 ± 1% AD-12603 100 9 100 ± 9%  −4 1 −4 ± 1% AD-12712 27 527 ± 5% 82 16 82 ± 16% AD-12604 15 2 15 ± 2% 94 13 94 ± 13% AD-12605 945 94 ± 5% 7 0 7 ± 0% AD-12713 61 10 61 ± 10%  41 8 41 ± 8% AD-12787 55 655 ± 6% 47 6 47 ± 6% AD-12834 92 16 92 ± 16%  8 2 8 ± 2% AD-12606 78 378 ± 3% 25 1 25 ± 1% AD-12714 63 6 63 ± 6% 42 5 42 ± 5% AD-12607 101 9101 ± 9%  −1 0 −1 ± 0% AD-12715 101 5 101 ± 5%  −1 0 −1 ± 0% AD-12788 8518 85 ± 18%  15 4 15 ± 4% AD-12835 95 9 95 ± 9% 6 1 6 ± 1% AD-12608 10313 103 ± 13%  −3 0 −3 ± 0% AD-12716 81 9 81 ± 9% 22 3 22 ± 3% AD-1278961 4 61 ± 4% 44 4 44 ± 4% AD-12836 103 11 103 ± 11%  −3 0 −3 ± 0%AD-12609 108 19 108 ± 19%  −9 2 −9 ± 2% AD-12717 94 17 94 ± 17%  7 1 7 ±1% AD-12610 88 9 88 ± 9% 14 2 14 ± 2% AD-12718 39 4 39 ± 4% 64 8 64 ± 8%AD-12611 38 6 38 ± 6% 69 12 69 ± 12% AD-12719 26 4 26 ± 4% 78 13 78 ±13% AD-12790 17 3 17 ± 3% 87 18 87 ± 18% AD-12837 22 4 22 ± 4% 81 16 81± 16% AD-12612 100 6 100 ± 6%  0 0 0 ± 0% AD-12720 73 6 73 ± 6% 28 3 28± 3% AD-12791 46 9 46 ± 9% 57 12 57 ± 12% AD-12838 97 15 97 ± 15%  3 1 3± 1% AD-12613 26 4 26 ± 4% 82 15 82 ± 15% AD-12721 10 1 10 ± 1% 94 12 94± 12% AD-12792 10 3 10 ± 3% 94 40 94 ± 40% AD-12839 22 3 22 ± 3% 81 1281 ± 12% AD-12614 15 5 15 ± 5% 94 38 94 ± 38% AD-12722 6 1  6 ± 1% 98 2698 ± 26% AD-12615 93 4 93 ± 4% 8 0 8 ± 0% AD-12616 95 4 95 ± 4% 5 0 5 ±0% AD-12723 73 7 73 ± 7% 30 3 30 ± 3% AD-12617 88 10 88 ± 10%  13 2 13 ±2% AD-12618 42 7 42 ± 7% 60 7 60 ± 7% AD-12724 21 5 21 ± 5% 89 32 89 ±32% AD-12619 95 7 95 ± 7% 6 1 6 ± 1% AD-12725 71 2 71 ± 2% 30 1 30 ± 1%AD-12793 54 7 54 ± 7% 48 7 48 ± 7% AD-12840 94 9 94 ± 9% 7 1 7 ± 1%AD-12620 106 7 106 ± 7%  −8 1 −8 ± 1% AD-12726 100 7 100 ± 7%  0 0 0 ±0% AD-12621 107 9 107 ± 9%  −7 1 −7 ± 1% AD-12727 47 4 47 ± 4% 60 8 60 ±8% AD-12794 40 8 40 ± 8% 67 20 67 ± 20% AD-12841 78 13 78 ± 13%  25 8 25± 8% AD-12622 16 4 16 ± 4% 92 29 92 ± 29% AD-12728 25 6 25 ± 6% 84 29 84± 29% AD-12795 23 3 23 ± 3% 86 20 86 ± 20% AD-12842 19 4 19 ± 4% 91 2091 ± 20% AD-12623 103 9 103 ± 9%  −3 0 −3 ± 0% AD-12729 84 8 84 ± 8% 172 17 ± 2% AD-12624 31 4 31 ± 4% 77 12 77 ± 12% AD-12730 18 1 18 ± 1% 853 85 ± 3% AD-12625 94 10 94 ± 10%  5 1 5 ± 1% AD-12731 57 4 57 ± 4% 48 448 ± 4% AD-12626 99 7 99 ± 7% 0 0 0 ± 0% AD-12732 82 5 82 ± 5% 20 1 20 ±1% AD-12627 80 6 80 ± 6% 22 2 22 ± 2% AD-12733 65 6 65 ± 6% 39 4 39 ± 4%AD-12628 81 6 81 ± 6% 21 2 21 ± 2% AD-12734 82 7 82 ± 7% 21 2 21 ± 2%AD-12629 113 11 113 ± 11%  −14 2 −14 ± 2% AD-12735 90 9 90 ± 9% 11 1 11± 1% AD-12796 92 8 92 ± 8% 9 1 9 ± 1% AD-12843 117 7 117 ± 7%  −19 1 −19± 1% AD-12630 124 3 124 ± 3%  −27 1 −27 ± 1% AD-12736 85 4 85 ± 4% 16 116 ± 1% AD-12797 52 1 52 ± 1% 53 1 53 ± 1% AD-12844 96 4 96 ± 4% 5 0 5 ±0% AD-12631 110 11 110 ± 11%  −12 1 −12 ± 1% AD-12737 115 13 115 ± 13% −17 2 −17 ± 2% AD-12632 106 2 106 ± 2%  −7 0 −7 ± 0% AD-12633 107 12 107± 12%  −8 1 −8 ± 1% AD-12738 88 5 88 ± 5% 14 1 14 ± 1% AD-12634 79 5 79± 5% 24 1 24 ± 1% AD-12739 69 8 69 ± 8% 35 6 35 ± 6% AD-12635 75 8 75 ±8% 25 6 25 ± 6% AD-12740 65 8 65 ± 8% 40 8 40 ± 8% AD-12798 56 4 56 ± 4%50 6 50 ± 6% AD-12845 74 6 74 ± 6% 30 3 30 ± 3% AD-12636 89 8 89 ± 8% 91 9 ± 1% AD-12741 31 4 31 ± 4% 78 14 78 ± 14% AD-12637 16 2 16 ± 2% 9314 93 ± 14% AD-12742 18 3 18 ± 3% 85 14 85 ± 14% AD-12799 18 4 18 ± 4%86 22 86 ± 22% AD-12846 15 2 15 ± 2% 89 14 89 ± 14% AD-12638 95 6 95 ±6% 5 0 5 ± 0% AD-12743 23 4 23 ± 4% 81 15 81 ± 15% AD-12800 14 1 14 ± 1%90 10 90 ± 10% AD-12847 90 12 90 ± 12%  10 2 10 ± 2% AD-12639 113 11 113± 11%  −15 2 −15 ± 2% AD-12744 42 4 42 ± 4% 60 7 60 ± 7% AD-12801 34 334 ± 3% 68 8 68 ± 8% AD-12848 114 3 114 ± 3%  −14 0 −14 ± 0% AD-12640 9611 96 ± 11%  4 1 4 ± 1% AD-12745 52 7 52 ± 7% 53 8 53 ± 8% AD-12802 74 974 ± 9% 29 4 29 ± 4% AD-12849 111 5 111 ± 5%  −12 1 −12 ± 1% AD-12641103 8 103 ± 8%  −3 0 −3 ± 0% AD-12803 94 13 94 ± 13%  6 1 6 ± 1%AD-12642 105 3 105 ± 3%  −6 0 −6 ± 0% AD-12746 100 9 100 ± 9%  0 0 0 ±0% AD-12643 33 4 33 ± 4% 74 10 74 ± 10% AD-12747 21 3 21 ± 3% 83 13 83 ±13% AD-12804 25 4 25 ± 4% 78 14 78 ± 14% AD-12850 28 4 28 ± 4% 75 11 75± 11% AD-12644 82 7 82 ± 7% 20 2 20 ± 2% AD-12748 25 4 25 ± 4% 78 14 78± 14% AD-12805 23 7 23 ± 7% 80 30 80 ± 30% AD-12851 61 7 61 ± 7% 41 5 41± 5% AD-12645 112 6 112 ± 6%  −14 1 −14 ± 1% AD-12749 86 10 86 ± 10%  162 16 ± 2% AD-12646 94 10 94 ± 10%  6 1 6 ± 1% AD-12750 93 11 93 ± 11%  71 7 ± 1% AD-12806 77 8 77 ± 8% 24 3 24 ± 3% AD-12852 96 4 96 ± 4% 5 0 5± 0% AD-12647 27 3 27 ± 3% 81 11 81 ± 11% AD-12751 29 6 29 ± 6% 74 19 74± 19% AD-12807 31 2 31 ± 2% 72 6 72 ± 6% AD-12853 26 3 26 ± 3% 78 11 78± 11% AD-12648 81 9 81 ± 9% 17 3 17 ± 3% AD-12649 92 9 92 ± 9% 8 1 8 ±1% AD-12752 71 9 71 ± 9% 30 5 30 ± 5% AD-12650 81 2 81 ± 2% 21 1 21 ± 1%AD-12753 57 1 57 ± 1% 48 1 48 ± 1% AD-12808 52 4 52 ± 4% 54 5 54 ± 5%AD-12854 77 5 77 ± 5% 26 2 26 ± 2% AD-12651 89 6 89 ± 6% 13 1 13 ± 1%AD-12754 88 7 88 ± 7% 12 1 12 ± 1% AD-12809 67 6 67 ± 6% 35 4 35 ± 4%AD-12855 88 10 88 ± 10%  12 2 12 ± 2% AD-12652 91 2 91 ± 2% 10 0 10 ± 0%AD-12755 40 3 40 ± 3% 67 6 67 ± 6% AD-12810 35 1 35 ± 1% 72 3 72 ± 3%AD-12856 75 8 75 ± 8% 28 4 28 ± 4% AD-12653 79 8 79 ± 8% 23 3 23 ± 3%AD-12756 17 5 17 ± 5% 86 27 86 ± 27% AD-12654 97 6 97 ± 6% 3 0 3 ± 0%AD-12811 74 5 74 ± 5% 27 2 27 ± 2% AD-12655 46 6 46 ± 6% 59 9 59 ± 9%AD-12757 14 0 14 ± 0% 89 2 89 ± 2% AD-12812 12 3 12 ± 3% 92 28 92 ± 28%AD-12857 35 7 35 ± 7% 70 17 70 ± 17% AD-12656 10 3 10 ± 3% 99 33 99 ±33% AD-12813 9 1  9 ± 1% 95 18 95 ± 18% AD-12657 108 1 108 ± 1%  −9 0 −9± 0% AD-12814 101 4 101 ± 4%  −1 0 −1 ± 0% AD-12658 98 9 98 ± 9% 2 0 2 ±0% AD-12659 83 4 83 ± 4% 18 1 18 ± 1% AD-12758 80 14 80 ± 14%  21 4 21 ±4% AD-12815 25 3 25 ± 3% 79 11 79 ± 11% AD-12858 67 4 67 ± 4% 35 2 35 ±2% AD-12660 95 11 95 ± 11%  8 3 8 ± 3% AD-12759 66 7 66 ± 7% 39 6 39 ±6% AD-12661 34 2 34 ± 2% 73 5 73 ± 5% AD-12760 10 3 10 ± 3% 94 30 94 ±30% AD-12816 12 4 12 ± 4% 92 37 92 ± 37% AD-12859 33 1 33 ± 1% 72 2 72 ±2% AD-12662 92 7 92 ± 7% 7 1 7 ± 1% AD-12817 91 11 91 ± 11%  10 2 10 ±2% AD-12663 99 10 99 ± 10%  3 1 3 ± 1% AD-12761 20 5 20 ± 5% 89 22 89 ±22% AD-12818 20 4 20 ± 4% 90 20 90 ± 20% AD-12860 93 11 93 ± 11%  8 1 8± 1% AD-12664 93 9 93 ± 9% 6 2 6 ± 2% AD-12665 94 8 94 ± 8% 10 1 10 ± 1%AD-12762 58 8 58 ± 8% 47 10 47 ± 10% AD-12819 49 6 49 ± 6% 58 9 58 ± 9%AD-12861 93 8 93 ± 8% 8 1 8 ± 1% AD-12666 30 5 30 ± 5% 76 18 76 ± 18%AD-12763 25 2 25 ± 2% 84 9 84 ± 9% AD-12667 65 10 65 ± 10%  38 7 38 ± 7%AD-12764 34 7 34 ± 7% 69 17 69 ± 17% AD-12668 34 4 34 ± 4% 73 10 73 ±10% AD-12765 13 3 13 ± 3% 91 22 91 ± 22% AD-12820 11 2 11 ± 2% 93 17 93± 17% AD-12862 19 4 19 ± 4% 87 22 87 ± 22% AD-12669 22 3 22 ± 3% 87 1287 ± 12% AD-12766 11 4 11 ± 4% 93 39 93 ± 39% AD-12670 45 3 45 ± 3% 61 561 ± 5% AD-12767 10 3 10 ± 3% 94 31 94 ± 31% AD-12821 12 1 12 ± 1% 92 1392 ± 13% AD-12863 41 4 41 ± 4% 64 8 64 ± 8% AD-12671 83 9 83 ± 9% 19 219 ± 2% AD-12822 74 7 74 ± 7% 29 3 29 ± 3% AD-12672 52 7 52 ± 7% 54 9 54± 9% AD-12768 28 3 28 ± 3% 81 12 81 ± 12% AD-12673 56 5 56 ± 5% 49 5 49± 5% AD-12769 36 2 36 ± 2% 72 5 72 ± 5% AD-12823 33 2 33 ± 2% 75 5 75 ±5% AD-12864 49 7 49 ± 7% 57 10 57 ± 10% AD-12674 90 9 90 ± 9% 11 1 11 ±1% AD-12770 45 6 45 ± 6% 61 9 61 ± 9% AD-12675 45 5 45 ± 5% 62 8 62 ± 8%AD-12676 47 6 47 ± 6% 59 9 59 ± 9% AD-12771 31 4 31 ± 4% 77 11 77 ± 11%AD-12824 31 3 31 ± 3% 77 10 77 ± 10% AD-12865 43 7 43 ± 7% 64 12 64 ±12% AD-12677 23 4 23 ± 4% 86 16 86 ± 16% AD-12825 22 4 22 ± 4% 87 16 87± 16% AD-12678 102 8 102 ± 8%  −2 0 −2 ± 0% AD-12772 101 13 101 ± 13% −1 0 −1 ± 0% AD-12826 99 1 99 ± 1% 1 0 1 ± 0% AD-12866 91 7 91 ± 7% 10 110 ± 1% AD-12679 81 8 81 ± 8% 21 2 21 ± 2% AD-12773 11 2 11 ± 2% 93 1993 ± 19% AD-12680 17 3 17 ± 3% 92 17 92 ± 17% AD-12774 15 2 15 ± 2% 8917 89 ± 17% AD-12827 11 2 11 ± 2% 93 18 93 ± 18% AD-12867 15 3 15 ± 3%91 22 91 ± 22% AD-12681 28 3 28 ± 3% 79 10 79 ± 10% AD-12775 8 1  8 ± 1%95 19 95 ± 19% AD-12682 43 6 43 ± 6% 63 9 63 ± 9% AD-12776 23 5 23 ± 5%80 19 80 ± 19% AD-12828 23 5 23 ± 5% 80 20 80 ± 20% AD-12868 25 4 25 ±4% 81 16 81 ± 16% AD-12683 17 2 17 ± 2% 91 15 91 ± 15% AD-12777 11 2 11± 2% 92 22 92 ± 22% AD-12829 12 1 12 ± 1% 92 11 92 ± 11% AD-12869 19 319 ± 3% 87 16 87 ± 16% AD-12684 87 12 87 ± 12%  14 2 14 ± 2% AD-12778 414 41 ± 4% 66 8 66 ± 8% AD-12685 35 1 35 ± 1% 72 1 72 ± 1% AD-12686 68 568 ± 5% 36 3 36 ± 3% AD-12779 58 5 58 ± 5% 47 5 47 ± 5% AD-12687 73 8 73± 8% 30 4 30 ± 4% AD-12780 62 8 62 ± 8% 42 7 42 ± 7% AD-12688 18 1 18 ±1% 91 4 91 ± 4% AD-12781 11 3 11 ± 3% 93 33 93 ± 33% AD-12689 96 4 96 ±4% 4 0 4 ± 0% AD-12782 45 7 45 ± 7% 58 10 58 ± 10% AD-12830 15 3 15 ± 3%89 19 89 ± 19% AD-12870 51 3 51 ± 3% 52 4 52 ± 4% AD-12690 93 6 93 ± 6%8 1 8 ± 1% AD-12783 36 3 36 ± 3% 66 7 66 ± 7% AD-12831 27 2 27 ± 2% 76 776 ± 7% AD-12871 81 18 81 ± 18%  21 5 21 ± 5%

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting theexpression of a human JC virus genome in a cell, wherein said dsRNAcomprises at least two sequences that are complementary to each otherand wherein a sense strand comprises a first sequence and an antisensestrand comprises a second sequence comprising a region ofcomplementarity which is substantially complementary to at least a partof a mRNA encoding JC virus, and wherein said region of complementarityis less than 30 nucleotides in length and wherein said dsRNA, uponcontact with a cell expressing said JC virus, inhibits expression ofsaid JC virus genome.
 2. The dsRNA of claim 1, wherein said firstsequence is selected from the group consisting of Tables 1a and b andsaid second sequence is selected from the group consisting of Tables 1aand b.
 3. The dsRNA of claim 1, wherein said dsRNA comprises at leastone modified nucleotide.
 4. The dsRNA of claim 2, wherein said dsRNAcomprises at least one modified nucleotide.
 5. The dsRNA of claim 3,wherein said modified nucleotide is chosen from the group of: a2′-O-methyl modified nucleotide, a nucleotide comprising a5′-phosphorothioate group, and a terminal nucleotide linked to acholesteryl derivative or dodecanoic acid bisdecylamide group.
 6. ThedsRNA of claim 3, wherein said modified nucleotide is chosen from thegroup of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modifiednucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modifiednucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, aphosphoramidate, and a non-natural base comprising nucleotide.
 7. ThedsRNA of claim 3, wherein said first sequence is selected from the groupconsisting of Tables 1a and b and said second sequence is selected fromthe group consisting of Tables 1a and b.
 8. The dsRNA of claim 6,wherein said first sequence is selected from the group consisting ofTables 1a and b and said second sequence is selected from the groupconsisting of Tables 1a and b.
 9. A cell comprising the dsRNA ofclaim
 1. 10. A pharmaceutical composition for inhibiting the expressionof a gene from the JC Virus in an organism, comprising the dsRNA ofclaim 1 and a pharmaceutically acceptable carrier.
 11. Thepharmaceutical composition of claim 10, wherein said first sequence ofsaid dsRNA is selected from the group consisting of Tables 1a and b andsaid second sequence of said dsRNA is selected from the group consistingof Tables 1a and b.
 12. The pharmaceutical composition of claim 10,wherein the pharmaceutically acceptable carrier comprises a lipid.
 13. Amethod for inhibiting the expression of a gene from the JC Virus in acell, the method comprising: (a) introducing into the cell the dsRNA ofclaim 1; and (b) maintaining the cell produced in step (a) for a timesufficient to obtain degradation of the mRNA transcript of a gene fromthe JC Virus, thereby inhibiting expression of a gene from the JC Virusin the cell.
 14. A method of treating, preventing or managingpathological processes mediated by JC virus expression comprisingadministering to a patient in need of such treatment, prevention ormanagement a therapeutically or prophylactically effective amount of thedsRNA of claim
 1. 15. A vector for inhibiting the expression of a genefrom the JC Virus in a cell, said vector comprising a regulatorysequence operably linked to a nucleotide sequence that encodes at leastone strand of the dsRNA of claim
 1. 16. A cell comprising the vector ofclaim 15.